SSC/ISTH classification of hemophilia A: can hemophilia center laboratories achieve the new criteria?

Summary.  To assess the practicality of the recent Scientific and Standardization committee (SSC) of the International Society on Thrombosis and Haemostasis (ISTH) recommendations in respect of the classification of hemophilia we distributed samples from three untreated subjects with hemophilia A to 91 UK hemophilia centers (HCs), comprising 20 comprehensive care centers (CCCs) and 71 HCs. Laboratories were requested to perform their routine factor (F)VIII:C assays and to classify the severity of hemophilia. Median values of < 1 U dL⁻¹ were obtained on two samples. However, for each of the two, approximately 30% of laboratories obtained results in the range 1–29 U dL⁻¹ and 1–33 U dL⁻¹ respectively. For one of these samples 17 laboratories diagnosed severe hemophilia despite obtaining FVIII:C levels in the range 1–5 U dL⁻¹. The median FVIII:C for the third sample was 5.8 U dL⁻¹ with a range of 1.5–36 U dL⁻¹. For this sample eight centers diagnosed severe hemophilia. Fifty-four laboratories obtained a result > 5 U dL⁻¹; 21 of these diagnosed mild hemophilia, 31 moderate hemophilia and two severe hemophilia. Results from CCCs were more accurate and more precise than those from HCs. Our results indicate a need for improved standardization of FVIII assays. In the UK there remains a lack of consensus in respect of the laboratory diagnostic criteria for the classification of hemophilia A.     

Break-through bleeding in relation to predicted factor VIII levels in patients receiving prophylactic treatment for severe hemophilia A

Summary.  Background: The role of prophylactic factor VIII (FVIII) to decrease hemophilic bleeding and arthropathy is well established. The rationale for this strategy is to convert patients with severe hemophilia A to a moderate clinical phenotype by reducing time spent with a FVIII level <1 IU dL⁻¹. Studies to date, however, have not demonstrated a strong link between FVIII level and the bleeding rate. Objectives : To assess the effect of FVIII level on break-through bleeding in patients with severe hemophilia A on prophylaxis. Patients/methods: This study analysed data from 44 patients aged 1–6 and 99 patients aged 10–65 years with severe hemophilia A (FVIII <1 IU dL⁻¹) who were treated with prophylactic FVIII as part of clinical studies assessing pharmacokinetics, safety and efficacy of a recombinant FVIII (Advate^®). Each patient had pharmacokinetic measurements and FVIII infusions recorded, and these were used to calculate time spent with a FVIII below 1, 2 and 5 IU dL⁻¹. Results: The data demonstrate that increasing time with a FVIII below 1 IU dL⁻¹ is associated with increased total bleeds and hemarthroses. Lack of adherence to the intended frequency of FVIII infusion was the most important determinant of low FVIII and increased bleeding. In children aged 1–6 years, the rate of bleeding was also influenced by FVIII half-life and clearance. Conclusions: These data have important implications for the management of patients with severe hemophilia.     

How factor VIIa works in hemophilia

Summary.  The influence of elevated platelet concentration and recombinant factor VIIa (rFVIIa) on thrombin generation at 5 pM tissue factor (TF) in a synthetic mixture corresponding to hemophilia B (SHB) and 'acquired' hemophilia B blood (AHBB) produced in vitro by an antifactor IX antibody was evaluated. (a) Thrombin generation in SHB and AHBB was delayed and reduced; (b) with 10 nM rFVIIa or 5× normal platelets (10 × 10⁸/mL) SHB and AHBB showed a slight increase in thrombin generation; (c) in the absence of TF, almost no thrombin generation was detected in SHB and AHBB in the presence of 10 nM rFVIIa and 10 × 10⁸/mL activated platelets (5× normal); (d) with TF, 10 nM rFVIIa and 3–5× normal nonactivated platelets (6–10 × 10⁸/mL), thrombin levels approaching normal values were attained. FVIIa appears to function effectively and locally by the combined effect of TF expression and platelet accumulation at the site of a vascular lesion.     

Liposomal dexamethasone effectiveness in the treatment of hypersensitivity pneumonitis in mice

Abstract. The effects of daily intranasal instillation of liposomal dexamethasone and free dexamethasone phosphate were compared in a murine model of hypersensitivity pneumonitis induced by Saccharopolyspora rectivirgula (formally known as Micropolyspora faeni ). After 3 weeks of antigen and liposome instillations, lung response was evaluated by bronchoalveolar lavage cell counts, lung index and histopathology. Systemic absorption was evaluated by measuring plasma adrenocorticotropic hormone (ACTH) level. Free dexamethasone phosphate induced a dose-dependent response with the maximal effect reached at 1 mgkg^-1. At 0.1 mgkg^-1, liposomal dexamethasone had a greater effect than free dexamethasone phosphate on bronchoalveolar cells ml^-1: 3.01 × 10⁵± 0.35× 10⁵ compared to 4.70×10⁵± 0.34 × 10⁵, and lung index: 1.22 ±0.10 compared to 1.86 ± 0.07. Effect on histopathology was similar. Plasma ACTH levels (pg ml^-1) were: 75.1 ± 14.0 for animals receiving antigen and free dexamethasone phosphate (0.2 mg kg^-1), and 149.7 ± 12.0 for animals receiving antigen and liposomal dexamethasone (0.2 mg kg^-1). In conclusion, liposome-incorporated dexamethasone is efficient in the treatment of experimental hypersensitivity pneumonitis and, contrarily to free dexamethasone phosphate, does not inhibit ACTH secretion.     

Non-classical anti-factor VIII C2 domain antibodies are pathogenic in a murine in vivo bleeding model

Summary.  Objective: The pathogenicity of anti-human factor (F) VIII monoclonal antibodies (MAbs) was tested in a murine bleeding model. Methods: MAbs were injected into the tail veins of hemophilia A mice to a peak plasma concentration of 60 n m , followed by injection of human B domain-deleted FVIII at 180 U kg⁻¹, producing peak plasma concentrations of ∼2 n m . At 2 h, blood loss following a 4-mm tail snip was measured. The following MAbs were tested: (i) 4A4, a type I anti-A2 FVIII inhibitor, (ii) I54 and 1B5, classical type I anti-C2 inhibitors, (iii) 2–77 and B45, non-classical type II anti-C2 inhibitors, and (iv) 2–117, a non-classical anti-C2 MAb with inhibitory activity less than 0.4 Bethesda Units per mg IgG. Results: All MAbs except 2–117 produced similar amounts of blood loss that were significantly greater than control mice injected with FVIII alone. Increasing the dose of FVIII to 360 U kg⁻¹ overcame the bleeding diathesis produced by the type II MAbs 2–77 and B45, but not the type I antibodies, 4A4, I54, and 1B5. These results were consistent with the in vitro Bethesda assay in which 4A4 completely inhibited both 1 U mL⁻¹ and 3 U mL⁻¹ FVIII, while there was 40% residual activity at saturating concentrations of 2–77 at either concentration of FVIII. Conclusions: For patients with an inhibitor response dominated by non-classical anti-C2 antibodies both the in vivo and in vitro results suggest that treatment with high-dose FVIII rather than bypassing agents may be warranted.     

Fatal transfusion-associated graft-versus-host disease in an immunocompetent recipient of a volunteer unit of red cells

BACKGROUND:  Transfusion-associated graft-versus-host disease (TAGVHD) is a lethal complication of transfusion of nonirradiated cellular blood components to a susceptible recipient.
CASE REPORT:  An 82-year-old man underwent cardiac surgery during which he received 6 units of red cells (RBCs) and a 6-unit pool of platelets (PLTs). He was discharged with a normal white blood cell (WBC) count and hemoglobin (Hb) level and a PLT count of 104 × 10⁹ per L. He was readmitted 2 weeks later with a diffuse erythematous rash, a sore throat, and difficulty swallowing. His WBC count was 2.1 × 10⁹ per L, his Hb level was 12.0 g per dL, and his PLT count was 131 × 10⁹ per L. The next day he had worsening cytopenias: WBC count, 1 × 10⁹ per L; Hb level, 10.9 g per dL; PLT count, 104 × 10⁹ per L. He also had diarrhea. A marrow biopsy showed a severe hypoplasia without evidence of malignancy. A skin biopsy showed Grade II GVHD. The patient worsened and despite aggressive therapy he expired on Postoperative Day 42. DNA-based HLA testing of the 12 blood donors was performed. One of the RBC donors was found to be homozygous for an HLA Class I and Class II haplotype in the patient.
CONCLUSION:  This is the first reported case in the United States of fatal TAGVHD from RBCs in an immunocompetent patient who received a randomly selected unit of RBCs from a donor who was homozygous for a shared HLA haplotype. The policy of selective irradiation should be reexamined.     

Factors influencing therapeutic efficacy and the host immune response to helper-dependent adenoviral gene therapy in hemophilia A mice

Summary.  Background : Adenoviral-based methods of gene therapy have been ineffective at providing sustained factor (F)VIII expression in outbred populations of large animal hemophilic models primarily due to the immunogenicity of these vectors. Improvements have been made in vector design leading to the development of the helper-dependent adenoviral (HD) system. Unfortunately, it remains unclear whether these modifications are sufficient to circumvent the induction of inhibitor formation associated with adenoviral gene transfer. Objective : To develop an HD vector capable of mediating sustained FVIII expression and to determine the variables that influence inhibitor development. Methods : HD vectors were constructed encoding the canine FVIII B-domain deleted transgene under the control of either the cytomegalovirus (CMV) promoter or a tissue-restricted hybrid element consisting of five HNF-1 binding sites, located upstream of the human FVIII proximal promoter. Inbred and outbred populations of hemophilic mice were treated, and monitored for vector-induced toxicity, therapeutic efficacy, and inhibitor formation. Results : When HD vectors utilizing the CMV promoter were administered, all hemophilic mice developed high levels of FVIII inhibitors. In contrast, vectors under the control of the HNF/FVIII element were capable of achieving sustained elevations of FVIII for over 6 months. Strain-specific differences were also observed, with outbred animals showing a greater propensity towards inhibitor development in response to treatment. Conclusions : HD vectors can be used to provide long-term FVIII expression in hemophilic animals, but treatment outcome and the induction of inhibitors is dependent on a number of variables including the transgene promoter, the vector dose, and the genetic background of the host.     

Mild hemophilia A with factor VIII assay discrepancy: using thrombin generation assay to assess the bleeding phenotype

Summary.  Introduction:  In some patients with mild hemophilia A, there are discrepancies between 1-stage (1-st) and 2-stage (2-st) factor VIII (FVIII) clotting assays, and also chromogenic assays for FVIII activity (FVIII:C). We examined whether thrombography could provide a better evaluation of the hemostatic status of these patients. Methods:  Two families with such discrepancies and markedly contrasting clinical histories were studied. Family X had no serious bleedings, in contrast to family Y. Sixty-one moderate/mild hemophiliacs without discrepancy and 15 healthy subjects served as controls. Calibrated automated thrombography was performed with platelet-rich plasma after one freeze-thawing cycle and low tissue factor concentration. Results:  The chromogenic FVIII:C levels were higher (0.90 ± 0.15 and 0.47 ± 0.13 IU mL⁻¹) than the 1-st clotting ones (0.14 ± 0.05 and 0.10 ± 0.05 IU mL⁻¹) in family X and Y, respectively ( P  < 0.001). Mean endogenous thrombin potential (ETP) was 1579 ± 359 n m  min⁻¹ and 1060 ± 450 for healthy controls and hemophilic controls, respectively. For members of family X, the ETP values were 1188, 1317 and 2277 n m  min⁻¹, whereas for those of family Y they ranged from 447 to 1122 n m  min⁻¹. Two novel missense point mutations were evidenced: p.Ile369Thr in family X and p.Phe2127Ser in family Y. In family X, we postulate that the mutation is responsible for a delayed but non-deleterious FVIII activation. Conclusions:  Our results suggest that the hemostatic phenotype assessed by thrombography may be clinically relevant in moderate/mild hemophilic patients with discrepant FVIII:C results.     

Elucidation of a novel epitope of a substrate-inducing monoclonal antibody against the serpin PAI-1

Summary.  Plasminogen activator inhibitor-1 (PAI-1) is the most important physiological inhibitor of plasminogen activators. Inhibition of PAI-1 constitutes a putative strategy for the prevention of cardiovascular disease. The monoclonal antibody MA-8H9D4 inhibits PAI-1 activity by inducing a substrate behavior in PAI-1. To identify the epitope, a rational approach was used to design various PAI-1 alanine mutants ( n  = 16) for evaluation of their affinity. PAI-1-R300A, PAI-1-Q303A and PAI-1-D305A had affinities for MA-8H9D4 of < 10⁵ M⁻¹, 2.0 × 10⁸ M⁻¹ and 2.5 × 10⁸ M⁻¹, respectively, whereas the affinity of wtPAI-1 is 3.3 × 10⁹ M⁻¹. The epitope on the axis of arginine 300, glutamine 303 and aspartic acid 305, located on the loop between α-helix I and β-strand 5A, demonstrates that MA-8H9D4 interferes with the final locking step in the serpin/proteinase interaction, thereby explaining its substrate inducing properties. The location of the epitope as well as the proposed mechanism of action is clearly different from that of other substrate inducing monoclonal antibodies against PAI-1. Elucidation of this novel epitope and the previously unidentified molecular mechanism opens new perspectives for the rational development of PAI-1-neutralizing compounds, as well as for the further exploration of synergistic effects between different PAI-1-inhibiting compounds.     

Platelet-delivered factor VIII provides limited resistance to anti-factor VIII inhibitors

Summary.  Background:  Gene therapy strategies directed at expressing factor (F)VIII in megakaryocytes has potential advantages in the treatment of hemophilia A. Among these is that platelet (p) FVIII may be effective in the presence of circulating anti-FVIII inhibitors. Objective:  We examined in a murine transgenic model whether pFVIII could correct the coagulation defect in FVIII^null mouse in the presence of circulating inhibitors. Methods:  FVIII^null mice that were transgenic for pFVIII (pFVIII/FVIII^null) were compared with FVIII^null mice receiving infused FVIII in a FeCl₃ carotid injury model in the presence of anti-FVIII inhibitors. Results:  After injury, pFVIII/FVIII^null mice were significantly more resistant to circulating inhibitors than after plasma FVIII correction in both an acute and chronic models of inhibitor exposure even although in the chronic model, significant amounts of inhibitor were stored within the platelets. Furthermore, bleeding in the pFVIII mice in the presence of inhibitors was not as a result of the development of thrombocytopenia. Conclusion:  In FVIII^null mice, pFVIII provides improved, but limited, protection in the presence of inhibitors of ∼6-fold greater Bethesda Units per mL relative to infused FVIII. Our findings differ from a recent report using a tail-clip exsanguination assay on the degree of efficacy of pFVIII in the presence of inhibitors. We propose that this difference in outcome is as a result of the sensitivity of the tail-vein exsanguination model to low levels of pFVIII.     

Faster onset of effect and greater efficacy of NN1731 compared with rFVIIa, aPCC and FVIII in tail bleeding in hemophilic mice

Summary.  Background : Recombinant factor VIIa (rFVIIa, Novoseven^®) is currently used to control bleeding in hemophiliacs with inhibitors. A new rFVIIa variant, NN1731, with increased activity on the surface of activated platelets, has demonstrated a more potent and faster onset of reactivity than rFVIIa in various in vitro models. The present study aimed to investigate whether this translates into greater efficacy and faster promotion of hemostasis in vivo . Method and results : In a severe tail-bleeding model in hemophilia A mice, NN1731 demonstrated significantly greater efficacy than rFVIIa, plasma-derived activated prothrombin complex concentrate (pd-aPCC, FEIBA^®) or FVIII (Refacto^®). Assessment of the blood loss over time showed that NN1731 significantly and dose-dependently reduced the blood loss in the first 5-min observation period, whereas the effect of rFVIIa, FVIII and pd-aPCC first became evident 5–10 min after injury. Conclusion : This study shows that NN1731 has a greater efficacy and faster resolution of bleeding in a severe bleeding model in hemophilia A mice compared with any of the other agents tested.     

Likelihood evaluation using 15 common short tandem repeat loci: a practical and simulated approach to establishing personal identification via sibling/parental assessments

BACKGROUND: In individual identification, often the only way to circumvent the lack of personal reference materials is to resort to a kinship analysis between the unidentified party (UP) and putative relatives.
STUDY DESIGN AND METHODS: Genotypes of 15 short tandem repeat loci in 20,000 families were simulated and the likelihood ratios (LRs) were calculated based on the frequencies of alleles in the Japanese population. These LR values illustrate the likelihood of whether an alleged kin is related to a UP. First, three sets of kinship were assessed: UP and one parent (or child); UP and two children; and UP and both parents. Next, an UP was compared with one to three alleged siblings (ASs). In the end, these calculations were applied to 21 actual cases where the identity of an individual lay in dispute.
RESULTS: When comparisons were made between a UP and both parents, or two children, the LR median dramatically increased (6.68 × 10¹¹ and 1.10 × 10⁸, respectively) in comparison to assessments involving an UP and one parent or child (48,900). Similarly, when two or three true siblings were made available for comparison, the LR was far greater (7.32 × 10⁶ and 4.50 × 10⁸, respectively) than in cases where only one sibling was available (9,250). Of the 21 actual cases these calculations were applied to, 1 case ended in exclusion with absolute certainty and the remaining 20 cases obtained high LRs.
CONCLUSION: As demonstrated, this system is extremely effective in cases of personal identification where both parents, two or more children, or two or more ASs are available for comparison.     

Plasma endothelin-1 levels during transient acute myocardial ischaemia in men: effects of coronary revascularization

The endothelium-derived peptide endothelin-1 (ET-1) was evaluated in 14 male patients [mean age 52.74 years (SEM 1.10)] affected by coronary artery disease during a bicycle electrocardiographic stress test and dipyridamole echocardiogram. Both tests were performed before and after coronary revascularization. Fourteen healthy male subjects served as controls [mean age 53.21 years (SEM 1.63)]. Baseline plasma endothelin-1 levels were higher ( P  < 0^.0001) in ischaemic patients [1^.81 pg mL⁻¹ (0^.15, n  = 14)] than in control subjects [0^.61 pg mL⁻¹ (0^.03, n  = 14)], but did not increase with exercise in both groups. Similar results were obtained with dipyridamole infusion. Endothelin-1 levels significantly decreased after coronary revascularization [before: mean 1^.81 pg mL⁻¹ (SEM 0^.15, n  = 14); after: mean 1^.16 pg mL⁻¹ (SEM 0^.11), P  < 0^.002], without changes in the peptide response to both tests. In conclusion, elevated plasma endothelin-1 concentrations were found in patients with stable angina compared with non-ischaemic subjects. No changes were observed during exercise or dipyridamole infusion in both groups. Coronary revascularization was followed by a significant decrease in plasma endothelin-1 levels.     

Elevated plasma levels of factor VIII in women with early recurrent miscarriage

Summary.  Aims : Inherited and acquired thrombophilia have been found to be associated with recurrent pregnancy loss. This paper examines whether or not elevated factor (F)VIII:C plasma levels, which have been demonstrated to be an independent risk factor for venous thromboembolism, are a risk factor for early recurrent miscarriages also. Patients and methods : Consecutive women referred to our clinic with a history of early recurrent abortion (at least three pregnancy losses before week 13 of gestation) were eligible for the study. Exclusion criteria were endocrine, immunological, anatomical and genetic causes of embryo demise, as well as any thrombophilic abnormality, either congenital or acquired, or a personal or familial history of venous thromboembolism. FVIII:C plasma levels were determined in 51 cases and in 51 controls matched for age, ethnicity and blood group. Results : The mean FVIII:C level in the control subjects was 106.8 IU dL⁻¹, compared with 128.2 IU dL⁻¹ in the patients group ( P  = 0.0002). Thirteen (25.5%) of the 51 patients had FVIII:C values exceeding the 90th centile of the control population (145 IU dL⁻¹), compared with four subjects in the control group (χ² = 4.52; P  = 0.033; odds ratio = 4.02, 95% confidence interval 1.09, 16.05). No cases with increase in FVIII:C levels attributable to an acute-phase reaction, as assessed by C-reactive protein plasma concentration, were found. Conclusions : We found FVIII:C levels significantly higher in women with early recurrent miscarriage compared with controls. This finding suggests a possible association between this thrombophilic condition and early reproductive failures.     

Enhancement of the enzymatic activity of activated coagulation factor IX by anti-factor IX antibodies

Summary.  Background:  Factor VIIIa (FVIIIa) binds to activated FIX and enhances the activation of FX by several orders of magnitude. Deficiency of FVIII causes the bleeding disorder hemophilia A and is treated by i.v. infusion of FVIII concentrates. Objectives:  To explore whether or not FVIII activity can be supplied by alternative molecules, e.g. molecules with FIXa-binding activity. Methods:  Conventional hybdridoma technology was used to discover antibodies exhibiting FVIII-like activity. Results:  We identified a series of antibodies specific for human FIX that mimicked the stimulatory effect of FVIIIa on FIXa. Upon binding to human FIXa, these antibodies enhanced the protease activity of FIXa towards its natural substrate FX about tenfold. A similar enhancement was also achieved with 5 p m FVIIIa (i.e . 16 mU mL⁻¹ or 1.6% activated FVIII). Procoagulant activity of these anti-FIXa antibodies was observed in model systems containing purified proteins as well as in plasma. Conclusion:  Our findings show that FVIII can, at least partially, be replaced by an unrelated molecule. Procoagulant antibodies might potentially aid the development of an FVIII substitute for hemophilia A treatment.     

The role of recombinant factor VIIa (FVIIa) in fibrin structure in the absence of FVIII/FIX

Summary.  Patients with hemophilia have an impaired thrombin generation and therefore form loose fibrin hemostatic plugs that are easily dissolved by fibrinolysis. This prevents maintained hemostasis in these patients, resulting in a severe bleeding disorder. Recombinant (F)VIIa has been shown to enhance thrombin generation on already thrombin-activated platelets in the absence of FVIII and FIX. An efficacy rate of 80–90% has been found in hemophilia patients with inhibitors against FVIII or FIX both in association with major surgery and in the treatment of serious bleedings. In a model measuring fibrin clot permeability in a platelet-containing system described by Blombäck et al . (1994) this was demonstrated to be dependent on the concentration of FVIII and FIX. The addition of rFVIIa in concentrations of 1.9, 4.8 and 9.6 µg mL⁻¹ normalized fibrin clot permeability. The concentration of 1.9 µg mL⁻¹ of rFVIIa normalized clot permeability in this system and the higher concentrations of rFVIIa added only slightly to the effect. No further decrease in clot permeability was found when rFVIIa in a concentration of 1.9 µg mL⁻¹ was added to a sample with a normal concentration (100%) of FVIII or FIX. Higher concentrations of rFVIIa added to the plasma containing 100% of FVIII or FIX induced only a slight further decrease of fibrin permeability constant, arguing against any unwanted effect of extra rFVIIa on clot permeability in the case of a normal hemostasis. Furthermore, the fibrin network was studied with 3D microscopy and the loose network found in the absence of FVIII or FIX increased in density with increasing FVIII or FIX concentrations. The addition of rFVIIa to FVIII- or FIX-deficient systems altered the network structure, making the fibers thinner and more tightly packed.     

Thrombosis triggered by severe arterial lesions is inhibited by oral administration of a glycoprotein IIb/IIIa antagonist

Platelet aggregation and thrombosis play an important role in the onset of acute coronary events. Regardless of the stimulus for activation, platelet thrombus formation is ultimately regulated through the IIb/IIIa receptor complex. The effects of oral administration of xemilofiban, a non-peptide mimetic of the RGDF sequence of the IIb/IIIa receptor complex, on thrombus formation were evaluated in a canine model. Xemilofiban significantly reduced platelet deposition on severely damaged arterial wall. Platelet deposition was reduced at both low (13 ± 1 from 56 ± 18 × 10⁶ platelets cm⁻²; P  < 0.05) and high (23 ± 2 from 111 ± 21 × 10⁶ platelets cm⁻²; P  < 0.01) shear rates. Platelet deposition was reduced to a monolayer as seen by electron microscopy (platelet–vessel wall interaction). Therefore, the availability of an orally active IIb/IIIa antagonist for chronic use may have significant value in preventing thrombus formation in those clinical situations associated with severe arterial injury, such as atherosclerotic plaque disruption.     

Higher infused CD34+ hematopoietic stem cell dose correlates with earlier lymphocyte recovery and better clinical outcome after autologous stem cell transplantation in non-Hodgkin's lymphoma

BACKGROUND: Lymphocyte recovery after autologous stem cell transplantation (ASCT) has been shown to be associated with positive clinical outcome in non-Hodgkin's lymphoma (NHL). This study sought to identify variables that affect lymphocyte recovery and survival after ASCT.
STUDY DESIGN AND METHODS: A retrospective analysis of outcomes in 97 consecutive patients with NHL who underwent ASCT in a single center from August 1999 to January 2008 was conducted.
RESULTS: A significant relationship was not observed between infused lymphocyte count and days to recovery of absolute lymphocyte count 500 × 10⁶/L or greater after ASCT (ALC500; r = 0.139, p = 0.176), but there was a significant inverse correlation between infused CD34+ cell count and days to ALC500 (r = −0.333, p = 0.001). Univariately, infused CD34+ cell count and recovery of ALC500 by 20th day after ASCT were significant predictors of survival. The median overall survival (OS) and event-free survival (EFS) were significantly longer in patients who received 8.2 × 10⁶ CD34+ cells/kg or more than in those who received fewer than 8.2 × 10⁶ CD34+ cells/kg (OS, not reached vs. 11.6 months, p = 0.001; EFS, not reached vs. 4.8 months, p = 0.003). Multivariate analysis confirmed that infused CD34+ cell count was an independent prognostic factor for OS (p = 0.017) and EFS (p = 0.002).
CONCLUSION: These data suggest that infused CD34+ cell count is predictive of kinetics of lymphocyte recovery after ASCT and is an independent prognostic factor for OS and EFS after ASCT in patients with NHL.     

Platelet count and the risk for thrombosis and death in the elderly

Summary.  Aim: Our aim was to examine the association between platelet count and the incidence of myocardial infarction, ischemic stroke, hemorrhagic stroke, venous thrombosis, and mortality. Methods and results: Platelet count was measured at baseline in 1989–1990 and at 3 years follow-up, or at baseline (for a newly recruited group) in 1992–1993 in 5766 community-dwelling individuals aged 65 years and older (mean age at baseline, 73 years). During 12–15 years of follow-up, there were 821 incident myocardial infarctions, 807 ischemic strokes, 161 hemorrhagic strokes, 159 venous thrombotic events, and 3413 participants died. Platelet count was not associated with the occurrence of myocardial infarction, ischemic or hemorrhagic stroke, venous thrombosis, or cardiovascular mortality. Non-cardiovascular mortality was higher among both participants with low and with high platelet count. Adjusted non-cardiovascular mortality rates for platelet counts below 100, 100–199, 300–399, and above 400 × 10⁹ L⁻¹ relative to the reference mortality rate in participants with platelet count values between 200 and 299 × 10⁹ L⁻¹ were 1.89 (1.21–2.96), 1.08 (0.98–1.20), 1.20 (1.06–1.37), and 1.47 (1.14–1.90), respectively. Conclusion: Platelet counts were not associated with vascular outcomes but low and high platelet counts were associated with non-cardiovascular mortality, including cancer mortality.     

Protein C inhibitor directly and potently inhibits activated hepatocyte growth factor activator

Background:  Hepatocyte growth factor (HGF) plays an important role in tissue repair and regeneration. HGF activator (HGFA), a factor XIIa-like serine protease, activates HGF precursor to HGF. The precursor of HGFA, proHGFA, is activated by thrombin generated at sites of tissue injury. It is known that protein C inhibitor (PCI), an inhibitor of activated protein C (APC), also inhibits thrombin–thrombomodulin (TM) complex. Objectives:  In the present study we evaluated the effect of PCI on thrombin-catalyzed proHGFA activation in the presence of TM, and on HGFA activity. Results:  PCI did not inhibit thrombin-TM-mediated proHGFA activation, but it directly inhibited activated HGFA by forming an enzyme inhibitor complex. The second-order rate constants ( m ⁻¹ min⁻¹) of the reaction between HGFA and PCI in the presence or absence of heparin (10 U mL⁻¹) were 4.3 × 10⁶ and 4.0 × 10⁶, respectively. The inhibition of HGFA by PCI resulted in a significant decrease of HGFA-catalyzed activation of HGF precursor. Exogenous HGFA added to normal human plasma formed a complex with plasma PCI, and this complex formation was competitively inhibited by APC in the presence of heparin, but very weakly in the absence of heparin. We also demonstrated using recombinant R362A-PCI that Arg362 residue of PCI is important for HGFA inhibition by PCI as judged from the three-dimensional structures constructed using docking models of PCI and HGFA or APC. Conclusion:  These observations indicate that PCI is a potent inhibitor of activated HGFA, suggesting a novel function for PCI in the regulation of tissue repair and regeneration.