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As proposed diagnostic criteria for DIC from the Japanese Society on Thrombosis and Hemostasis has been approved and revised, the contents and changes are informed.

article i nfo Introduction: We assessed the safety and effectiveness of recombinant soluble thrombomodulin (thrombomodulin alfa, TM-α) in the treatment of disseminated intravascular coagulation (DIC) in a post-marketing surveillance. Methods: The cases of 3548 patients with DIC caused by infection (n = 2516, Infection-DIC) or hematological malignancy (n = 1032, Hemat-DIC) were analyzed and compared to the results of a phase III (P-III) study. Results: The DIC scores were significantly decreased in the Infection-DIC and Hemat-DIC groups with TM-α treat- ment (both P b 0.001). The incidences of critical bleeding adverse drug reactions (ADRs) in the Infection-DIC and Hemat-DIC groups were 2.6% and 2.4%, and the survival rates were 64.1% and 70.7%, respectively. Patients with DIC were subcategorized into three groups (Infection-DIC-1 or Hemat-DIC-1, P-III criteria-matched patients; Infection-DIC-2 or Hemat-DIC-2, P-III criteria-non-matched patients treated solely with TM-α ;a nd Infection-DIC-3 or Hemat-DIC-3, P-III criteria-non-matched patients treated with TM-α and other concomitant anticoagulants). Subcategory analysis revealed that the incidences of critical bleeding ADRs of Hemat-DIC-2 and Hemat-DIC-3 were significantly higher and their survival rates were significantly lower than those of Hemat-DIC-1. By multivariate analysis in Hemat-DIC, younger age (odds ratio: 2.629, P = 0.0033) and pre- existing bleeding (odds ratio: 2.044, P = 0.019) were found to affect bleeding ADRs and the severity of underlying disease was the most important factor for survival rate (odds ratio: 0.288, P b 0.001). Conclusions:This surveillance provided real-world data for the safety and effectiveness of TM-α in the treatment of Infection-DIC and Hemat-DIC in general practice settings.

Disseminated intravascular coagulation (DIC) is a serious disease that, in the presence of underlying disease, causes persistent, generalized, marked coagulation activation. Early treatment based on an appropriate diagnosis is very important for improving patients’ prognosis, to which end diagnostic criteria play a key role. Several criteria have been proposed, but each has its strengths and weaknesses, and improved criteria are needed. Widespread use of coagulofibrinolytic markers has elucidated that the pathology of DIC differs greatly as a function of the underlying disease. Thus, discriminating use of DIC diagnostic criteria that take underlying diseases into account is important. DIC diagnostic criteria that are well known in Japan include the Japanese Ministry of Health and Welfare’s old DIC diagnostic criteria (JMHW criteria), the International Society on Thrombosis and Haemostasis’s DIC diagnostic criteria (ISTH criteria), and the Japanese Association for Acute Medicine’s acute-stage DIC diagnostic criteria (JAAM criteria). Those criteria have their respective drawbacks: the sensitivity of the ISTH criteria is poor, the JAAM criteria cannot be applied to all underlying diseases, and the JMHW criteria have poor sensitivity in the case of infections, do not use molecular markers, and result in misdiagnosis. The Japanese Society on Thrombosis and Hemostasis’s newly proposed provisional draft DIC diagnostic criteria (new criteria) use diagnostic criteria classifications of “hematopoietic disorder type”, “infectious type”, and “basic type” based on the underlying pathology. For the hematopoietic disorder type the platelet count is omitted from the score, while for the infectious type, fibrinogen is omitted from the score. Also, points are added if the platelet count decreases with time. In the new criteria, molecular markers and antithrombin activity have been newly included, and as a countermeasure for misdiagnosis, 3 points are deducted if there is liver failure. In this paper, we discuss various problems encountered with DIC diagnosis, and we describe the new criteria together with the events that led to their creation. These new diagnostic criteria take into account the underlying diseases of wide area, and we expect that they will serve clinicians well due to the above adaptations and improvements.

This retrospective analysis investigated the prognostic value of monitoring the response to imatinib using peripheral blood (PB) samples and the impact of the response on outcome in 133 patients with chronic myeloid leukemia (CML). We divided the response into 3 categories according to the results of neutrophil (N)-FISH and BCR-ABL transcript levels in PB; more than a 3-log reduction [major molecular response (MMR)], between a 2-log and 3-log reduction or negative with N-FISH [complete cytogenetic response equivalent (CCyRe)], N-FISH positive or less than a 2-log reduction (non-CCyRe). The median follow-up was 5.46 years. At 5 years, the overall survival (OS) rate and progression-free survival (PFS) rate were 94.4 and 92.0%, respectively. The estimated rate of the CCyRe and MMR were 81.7 and 67.1%, respectively. 106 patients achieving the CCyRe had significantly better OS and PFS than 27 patients without achieving the CCyRe. Patients with MMR had significantly better survival free from death, progression, imatinib withdrawal and a loss of the CCyRe, than patients whose response level remained in the CCyRe without achieving MMR until 18 months. Our observation suggests that the response level of the CCyRe on PB serve as a prognostic indicator, and achieving MMR provides stable long-term survival.

Acute intravascular haemolysis and renal failure developed while a patient was taking triamterene. A direct antiglobulin test with a polyvalent reagent was positive. Serum caused agglutination of normal red cells in the presence of triamterene and caused an increase of partial haemolysis of both trypsin-treated erythrocytes and red cells from a patient with paroxysmal nocturnal haemoglobinuria (PNH) in the presence of complement. From the results of antibody-neutralization test and treatment with 2-mercaptoethanol, the presence of IgM antibody with Λ light chain could be demonstrated. The triamterene seemed to bind strongly to the red cells in vitra but in vivo there was no detectable adsorption to red cells. Haptenic inhibition was not demonstrated. From these results, it was assumed that this antibody caused acute intravascular haemolysis by immune complex mechanism. The antibody was found to cross-react with methotrexate which has a structure similar to that of triamterene.

We identified 3 mutations in the factor V (FV) gene (F5) associated with FV deficiency in 3 unrelated Japanese patients. Patient 1 had severe bleeding symptoms (plasma FV activity

Introduction: Vascular endothelial cell damage plays an important role in microvascular thrombogenesis. In vivo administration of cyclosporin A or mitomycin C sometimes results in thrombotic microangiopathy in patients. Materials and methods: The effects of cyclosporin A, mitomycin C and/or prednisolone on the cell cycle in cultured human umbilical vein endothelial cells were investigated to evaluate drug-induced endothelial cell damage and the protective effect of prednisolone on endothelial cells against the damage by cyclosporin A or mitomycin C in vitro. Results: The addition of cyclosporin A to cultures caused proliferation arrest in the G1-phase in a dose-dependent manner, while mitomycin C inhibited DNA synthesis, which resulted in cell cycle arrest and inhibition of BrdUrd incorporation in the S-phase. The administration of prednisolone also caused cell cycle arrest in the G1 by itself, and protected the cells from the damage caused by mitomycin C. The inhibitory effects of cyclosporin A and prednisolone on the cell cycle were reversible, while mitomycin C was not. The highly phosphorylated retinoblastoma protein expressed in human umbilical vein endothelial cells decreased in the presence of mitomycin C. Soluble thrombomodulin levels in the culture supernatant were elevated by the addition of cyclosporin A. Conclusion: These effects of the drugs may cause the cell cycle arrest and the prolonged repair of damaged endothelial cells in patients. D 2004 Elsevier Ltd. All rights reserved.

A randomized prospective double-blind trial was performed to compare the safety and efficacy of human activated protein C (APC) and unfractionated heparin for the treatment of disseminated intravascular coagulation (DIC). One hundred thirty-two patients with DIC were enrolled in this study: 63 patients received APC (12.5 U [2.5 microg]/kg body wt per hour) and 69 patients received heparin (8 U/kg body wt per hour) by intravenous infusion for 6 days. Forty-nine APC-treated patients and 55 heparin-treated patients were evaluated for efficacy, and 52 APC-treated patients and 55 heparin-treated patients were evaluated for safety. The 2 groups were similar with respect to sex, age, body weight, underlying diseases, and coagulation/fibrinolysis parameters before treatment. Aggravation of bleeding was seen after treatment in 8 patients receiving heparin, but in none of the patients receiving APC. The number of patients who showed alleviation of bleeding was significantly higher in the APC group than the heparin group (P = .009). The effects on DIC-related organ dysfunction were not significantly different between the 2 groups. Fibrinogen-fibrin degradation products, D-dimer, thrombin-antithrombin complex (TAT), and plasmin-plasmin inhibitor complex (PIC) were all significantly decreased by treatment in both groups. Fibrinogen, protein C, and antithrombin were significantly increased in the APC group, whereas only protein C was significantly increased in the heparin group. Platelet count in the nonleukemic group was significantly increased in those patients receiving APC but not increased in those patients receiving heparin. Improvement of coagulation/fibrinolysis was assessed by scoring 4 parameters (soluble fibrin monomers, D-dimer, TAT, and PIC), and the results indicated that the APC group showed significantly greater improvement than the heparin group (P = .046). There was, however, no significant difference in the rate of complete recovery from DIC between the 2 groups. The rate of death from any cause within 28 days after treatment was 20.4% in the APC group, significantly lower than the 40% death rate observed in the heparin group (P.05). There were no severe adverse events in either group. These results suggest that APC in a relatively small dosage can improve DIC more efficiently than can heparin, without increasing bleeding, and may be a better alternative.

Recombinant human soluble thrombomodulin (thrombomodulin α [TM-α]) has been marketed as a novel anticoagulant for disseminated intravascular coagulation (DIC) in Japan since 2008. Postmarketing surveillance (PMS) has been conducted since its approval. As effectiveness and safety were not previously determined in pediatric patients, this study evaluated PMS data and examined the usefulness of TM-α in treating pediatric DIC. After excluding newborn infants, data for 210 pediatric patients were analyzed and compared to 3786 adult patients. The day after the last TM-α administration, DIC had resolved in 58.5% of the patients. At 28 days after the last TM-α administration, the survival rate was 71.6%. Nineteen episodes of adverse drug reactions were observed in 11 patients but no significant differences were noted for effectiveness and safety. Although this study was limited by its retrospective design, including selection biases and no limitation on concomitant use of other anticoagulants, TM-α appears to be useful for the treatment of DIC in both pediatric and adult patients.

A novel BβAsn-160 (TAA) to Ser (TGA) substitution has been identified in fibrinogen Niigata derived from a 64-year-old asymptomatic woman, who is heterozygotic for this abnormality. The mutation creates an Asn-X-Ser–type glycosylation sequence, and a partially sialylated biantennary oligosaccharide was linked to the BβAsn-158 residue. The functional abnormality was attributed to delayed lateral association of normally formed double-stranded protofibrils based on normal cross-linking of fibrin γ-chains and tissue-type plasminogen activator-catalyzed plasmin generation by polymerizing fibrin monomers. Enzymatic removal of all the N-linked oligosaccharides from fibrinogen Niigata accelerated fibrin monomer polymerization that reached the level of untreated normal fibrin monomers, but the thrombin time was prolonged from 18.2 seconds to 113 seconds (normal: 11.2 seconds to 8.9 seconds). By scanning electron micrographic analysis, Niigata fibrin fibers were found to be more curvilinear than normal fibrin fibers. After deglycosylation, Niigata fibers became straight being similar to untreated normal fibrin fibers, whereas normal deglycosylated fibrin appeared to be less-branched than untreated normal or deglycosylated Niigata fibrin. Although normal and Niigata fibrins were similar to each other in permeation and compaction studies, deglycosylated normal and Niigata fibrins had much higher permeability and compaction values, indicating that deglycosylation had brought about the formation of more porous networks. The enzymatic deglycosylation necessitates an Asn to Asp change at position Bβ-158 that is responsible for reducing the fiber thickness because of either local repulsive forces or steric hindrance in the coiled-coil region.

We experienced a case of Burkitt's lymphoma showing an unusual surface phenotype, CD5 expression, at an early stage of the disease. Initially, this patient showed massive abdominal para-aortic lymph node swelling which rapidly developed into leukemic change. Based on the clinical course and cytogenetic features of lymphoblasts in the bone marrow, which showed t(8;14) and c-myc gene rearrangement, the patient was diagnosed with Burkitt's lymphoma. Combination chemotherapy induced short-term remission, but central nervous system (CNS) involvement developed, followed by a regrowth of lymphoma cells in the bone marrow. The bone marrow at the end stage showed monotonous expansion of large cells with conspicuous vacuolation in the basophilic cytoplasm. The initial lymphoma cells showed pan-B markers and were CD5 positive but weakly CD10 positive; however, the lymphoma cells obtained from the bone marrow at the terminal stage did not express CD5. The chromosomal t(8;14) was seen, and identical rearrangement of immunoglobulin heavy chain joining gene and c-myc gene were detected by Southern blot analysis in the bone marrow lymphoblasts throughout the clinical course. This case is evidence that remarkable transformation of CD5-positive lymphoblasts to CD5-negative lymphoblasts occurred in an identical clone of Burkitt's lymphoma.

A patient presented with chronic large granular lymphocyte leukemia associated with chronic active Epstein-Barr virus infection (CAEBV). Cell cycle analysis revealed a minimal growth compatible with chronic lymphocytic leukemia After 5 months of treatment, the patient died from acute transformation of the leukemia. Cell harvested during chronic phase were analyzed for sensitivity to interleukin 2 (IL-2) and interferon alpha (IFN alpha) in vitro by means of surface phenotyping and cell cycle assay. IL-2 induced remarkable growth of the cells, whereas IFN alpha did not confer a growth advantage. Since IFN alpha was expected to have no growth induction effect on the leukemia cells, it was administered to the patient to treat the CAEBV.

To evaluate the clinical implications of soluble thrombomodulin and lipoprotein (a) [Lp(a)] in patients with cerebral thrombosis, these parameters were measured in the plasma of 28 patients with cerebral thrombosis within 3 days of onset, 36 with cerebral thrombosis more than 1 month after onset, six with cerebral hemorrhage more than 3 months after onset and 37 healthy volunteers. In the patients with chronic-phase cerebral thrombosis, the thrombomodulin and Lp(a) levels were significantly higher and the total cholesterol level was significantly lower than in the normal group, while the patients with acute-phase cerebral thrombosis had significantly lower total cholesterol levels. The plasma level of Lp(a) in acute-phase cerebral thrombosis, but not that of thrombomodulin, was significantly higher in thromboses located in the cortex area and in patients with recurrent attacks than in the normal controls. There were no significant differences in thrombomodulin, Lp(a) or total cholesterol levels between the chronic-phase cerebral hemorrhage and normal groups. These findings support the hypothesis that Lp(a) plays a part as a risk factor in cerebral thrombosis, especially in patients with a cortex area thrombosis and in patients with a recurrent attack. The high levels of thrombomodulin in the chronic-phase cerebral thrombosis group suggests the presence of continuous endothelial cell damage.

Platelet-type von Willebrand disease (vWD) is a congenital bleeding disorder characterized by heightened ristocetin-induced platelet aggregation caused by abnormally high affinity between the platelet membrane glycoprotein (GP) Ib/IX complex and von Willebrand factor (vWF ). Two distinct point mutations, Gly233 to Val and Met239 to Val, have been reported in GPIbα. We have constructed a recombinant GPIbα fragment containing the latter mutation, Met239 to Val (M239V) and characterized the mutant molecule using two methods, ie, interaction between soluble vWF and immobilized M239V and inhibition of platelet aggregation by purified soluble M239V. Spontaneous binding (ie, binding without any inducers) was observed between 125I-vWF and immobilized M239V but not between 125I-vWF and immobilized wild-type (WT) GPIbα. The addition of low concentrations of ristocetin (0.2 mg/mL) induced specific 125I-vWF binding to immobilized M239V, but not to WT GPIbα. At high concentrations of ristocetin (1.2 mg/mL), both WT GPIbα and M239V specifically bound to 125I-vWF. Thus, M239V reproduced the unique functional abnormality of the GPIb/IX complex in platelet-type vWD. Moreover, the purified soluble M239V inhibited platelet aggregation induced by low concentration of ristocetin (0.3 mg/mL) in platelet-rich plasma from a patient having Met239 to Val mutation, whereas purified WT did not. These results provide direct evidences that the reported point mutation is the responsible molecular basis of this disorder.

Platelet-type von Willebrand disease (vWD) is a congenital bleeding disorder characterized by heightened ristocetin-induced platelet aggregation caused by abnormally high affinity between the platelet membrane glycoprotein (GP) Ib/IX complex and von Willebrand factor (vWF ). Two distinct point mutations, Gly233 to Val and Met239 to Val, have been reported in GPIbα. We have constructed a recombinant GPIbα fragment containing the latter mutation, Met239 to Val (M239V) and characterized the mutant molecule using two methods, ie, interaction between soluble vWF and immobilized M239V and inhibition of platelet aggregation by purified soluble M239V. Spontaneous binding (ie, binding without any inducers) was observed between 125I-vWF and immobilized M239V but not between 125I-vWF and immobilized wild-type (WT) GPIbα. The addition of low concentrations of ristocetin (0.2 mg/mL) induced specific 125I-vWF binding to immobilized M239V, but not to WT GPIbα. At high concentrations of ristocetin (1.2 mg/mL), both WT GPIbα and M239V specifically bound to 125I-vWF. Thus, M239V reproduced the unique functional abnormality of the GPIb/IX complex in platelet-type vWD. Moreover, the purified soluble M239V inhibited platelet aggregation induced by low concentration of ristocetin (0.3 mg/mL) in platelet-rich plasma from a patient having Met239 to Val mutation, whereas purified WT did not. These results provide direct evidences that the reported point mutation is the responsible molecular basis of this disorder.

Introduction Patients with acute promyelocytic leukemia (APL) can develop disseminated intravascular coagulation (DIC) that results in life-threatening hemorrhagic complications. Studies regarding the safety and efficacy of thrombomodulin alfa (TM-α; recombinant human soluble thrombomodulin) in patients with APL and DIC are limited. Materials and methods A retrospective evaluation was performed on a cohort of 172 patients with APL from an open-label, multicenter, post-marketing surveillance study of TM-α. Results Of the 172 patients, 31 were relapse/refractory APL patients, and 141 were newly diagnosed APL patients. Within the first 30 days, 24 patients (14.0%) died, and six of those deaths (3.5%) were due to hemorrhage. In total, 12 patients (7.0%) had severe hemorrhagic complications. Both the early death rate due to hemorrhage as well as the severe hemorrhage rate did not exceed those in some recent population-based studies of patients with APL. Forty-nine patients received TM-α prior to the initiation of antileukemic treatment, and one patient experienced hemorrhagic early death (ED), suggesting that early TM-α treatment appeared to result in a reduction in the hemorrhagic ED rate. Moreover, TM-α improved coagulopathy regardless of concomitant all-trans retinoic acid treatment. Conclusions This study confirmed the safety and efficacy of TM-α in daily clinical practice for patients with APL and DIC. TM-α appeared to reduce hemorrhagic early deaths due to DIC in patients with APL who were receiving antileukemic treatment.

In clinical practice, occasionally some patients show dissociated values of fibrinogen/fibrin degradation products (FDP) and D-dimer (cross-linked fibrin degradation products). In an attempt to assess the frequency, clinical backgrounds, and hemostatic states of these cases, FDP and D-dimer were simultaneously measured together with other hemostatic parameters in 371 samples from patients with various diseases. As a whole, FDP were elevated in parallel with the progress of activation of blood coagulation and fibrinolysis. However, in patients with elevated FDP and/or D-dimer, 11.5% of samples showed relatively lower D-dimer values than those expected from FDP levels, and these were regarded as an apparently dissociated group. In the dissociated group, activation of coagulation and fibrinolysis occurred to a lesser extent than others. Analysis of these samples suggested that the possible reasons for the dissociation between FDP and D-dimer values were accelerated fibrinogenolysis with or without secondary fibrinolysis, accelerated fibrinogenolysis by non-plasmic proteinases, elevated soluble fibrin, and possibly false-positive FDP levels due to unclottable fibrinogen remaining in the serum samples. In practice, simultaneous measurements of FDP and D-dimer are useful for more accurate estimation of hyperfibrinolytic states.

Genomic DNA was studied from four patients with platelet-type von Willebrand disease (vWD) from two Japanese families previously reported. The entire coding region of platelet glycoprotein (GP) Ib alpha, a component of the platelet receptor for von Willebrand factor (vWF), was examined by polymerase chain reaction (PCR) followed by direct DNA sequence analysis. A single point mutation was found in all patients resulting in substitution of Val (GTG) for Met (ATG) at residue 239 of GPIb alpha. All patients were heterozygous for the mutation, whereas none of the unaffected family members had an amino acid substitution at residue 239. Because the nucleotide substitution destroys an NIa III restriction site on GPIb alpha, PCR products were subjected to digestion with this enzyme; DNA fragments from both normal and mutant alleles were detected in all affected individuals. In allele- specific PCR, DNA was amplified from patients' genomic DNA using either adenine- or guanine-containing primers, whereas only adenine-containing primer successfully amplified DNA from normal individuals. Cloning of amplified DNA into bacteriophage M13mp19 and subsequent DNA sequence analysis confirmed the mutation in these families. The absence of the amino acid substitution at residue 239 of GPIb alpha in the normal individuals tested, together with the linkage of this substitution to the phenotypic expression of disease in these two families and in a family recently described suggest that this amino acid change is a molecular basis for platelet-type vWD, and the substitution may produce a quite similar phenotype to the one reported previously (Gly to Val at residue 233 of GPIb alpha).

Recently it has been shown that tissue factor (TF), an important trigger for initiating blood coagulation, is present in the circulating plasma. In order to assess the clinical implications of TF in plasma, plasma concentration of TF was quantitated in 65 patients with disseminated intravascular coagulation (DIC). The mean concentration of plasma TF was elevated in patients with DIC at presentation as compared with healthy subjects (446 ± SD 536 pg/ml vs. 138 ± 51 pg/ml, P < 0.001). Abnormally high levels were found only in 46.2% of the patients, predominantly in patients with non-hematological solid tumors and acute leukemia. Plasma TF did not correlate with hemostatic markers of DIC such as thrombinantithrombin III complex, prothrombin fragment 1 + 2, plasmin-α2-plasmin inhibitor complex, FDP, D-dimer, or fibrinogen. Serial determinations of plasma TF demonstrated that plasma TF changes roughly in parallel with the course of DIC in most patients with elevated TF at presentation of DIC. These findings suggest that plasma TF is potentially valuable for monitoring the progress of DIC in a limited population of patients. © 1994 Wiley-Liss, Inc.

Plasma levels of urokinase-type plasminogen activator (u-PA) were measured with an enzyme-linked immunosorbent assay in patients with leukemias. As compared with healthy subjects (0.73 +/- SD 0.17 ng/ml), plasma u-PA antigen level was markedly elevated in patients with acute promyelocytic leukemia (APL) (1.76 +/- 0.89 ng/ml) at disease onset. Mean u-PA concentrations in patients with other acute nonlymphoblastic leukemia (0.57 +/- 0.51 ng/ml), acute lymphoblastic leukemia (0.77 +/- 0.82 ng/ml) and chronic myelocytic leukemia in blastic crisis (1.30 +/- 1.35 ng/ml) were not significantly elevated, but some of them showed an elevation of plasma u-PA. Plasma u-PA values were correlated with some of the fibrinolytic parameters such as FDP and D-dimer. Plasma u-PA antigen was decreased after the administration of antileukemic drugs in patients with APL. These results suggest that the coagulopathy in patients with various leukemias may in part be associated with u-PA release from the leukemic cells, especially in patients with APL.

Serum thrombomodulin (sTM) is an endothelial cell marker which would reflect the endothelial damage. In order to examine whether some antiplatelet agents decrease the endothelial damage in diabetic patients, sTM concentrations were measured by enzyme-linked immunosorbent assay before and after oral administration of a daily 100 mg of cilostazol for 4 weeks in 9 diabetics or a daily 81 mg of acetyl salicylic acids (ASA) for 4 weeks in 8 diabetics. Basal concentrations of sTM were elevated in most of these patients as compared with healthy subjects. The sTM concentrations were decreased after administration of cilostazol from 28.1 +/- 7.1 ng/ml to 23.6 +/- 5.4 ng/ml (p0.01), and after ASA from 30.7 +/- 10.9 ng/ml to 27.9 +/- 11.6 ng/ml (p0.05). These results suggest that such drugs can decrease the endothelial damage, resulting in the reduced risk of diabetic vascular complications.

Plasma levels of prothrombin fragment 1 + 2 (F1 + 2) and thrombin-antithrombin III complex (TAT) are thought to be specific indicators of thrombin generation. To assess whether these two parameters behave similarly in vivo, we compared the plasma levels of F1 + 2 with TAT in 41 patients with disseminated intravascular coagulation (DIC). Both F1 + 2 and TAT were markedly elevated in patients with DIC compared to healthy subjects. Although a positive correlation was found between F1 + 2 and TAT (r = 0.585, P < 0.001) there was a large scatter among individuals. In addition, plasma concentrations of TAT were much lower than F1 + 2. The correlation between the TAT/F1 + 2 ratio and antithrombin III was weak (r = -0.268, P = 0.09). The TAT/F1 + 2 ratio was positively correlated with TAT (r = 0.481, P = 0.002), indicating that the difference in molar concentrations between F1 + 2 and TAT decreased as the TAT value increased. Serial determinations of these parameters showed that plasma TAT values changed roughly in parallel with F1 + 2 in the majority of patients. Although further studies are required to further clarify the observed differences between F1 + 2 and TAT, both parameters would be equally useful for the precise detection of haemostatic activation in patients with DIC.

Plasma levels of von Willebrand factor (vWf) are frequently elevated in patients with disseminated intravascular coagulation (DIC). To investigate the qualitative abnormalities of vWf and the possibility of its ex vivo modification in DIC, we analysed the multimeric composition of vWf in citrated plasma from 15 patients with DIC in the presence or absence of serine protease inhibitors (aprotinin and soybean trypsin inhibitor) and/or cysteine protease inhibitors (leupeptin, N-ethylmaleimide and EDTA). The proportion of large vWf multimers in plasma prepared in the presence of cysteine protease inhibitors was higher than those without such inhibitors. The addition of serine protease inhibitors during the preparation of plasma had no effect on the relative amounts of large multimers. The relative proportion of large multimers in plasma prepared without inhibitors and the difference between plasmas prepared with and without cysteine protease inhibitors correlated with plasma plasmin-alpha 2-plasmin inhibitor complex values, but not with other plasma or serum markers of DIC (platelet count, fibrinogen, FDP, D-dimer or thrombin-antithrombin III complex). We conclude that ex vivo proteolysis of plasma vWf occurs frequently in patients with DIC and cysteine protease inhibitors can protect this degradation.

A rare association of congenital afibrinogenemia and hereditary protein C deficiency is described in a 37-year-old female who suffered from ischemic necrosis in the left first toe. The diagnosis of afibrinogenemia was assessed by the absence of fibrinogen in clotting and immunological assays. The diagnosis of hereditary heterozygous type I protein C deficiency was based on the evidence of proportional decreases of activity and antigen of plasma protein C in the propositus, her mother, and two maternal aunts.

One approach to improving the outcome of allogeneic hematopoietic stem-cell transplantation for acute lymphoblastic leukemia (ALL) is to intensify the pretransplant conditioning regimen without increasing toxicity. We used an intensified conditioning regimen consisting of high-dose cytosine arabinoside (3 g/m(2) twice daily i.v. for 3 consecutive days, total six doses), high-dose etoposide (1 g/m(2) once daily i.v. during the first 2 days) and total body irradiation (TBI) (HDACE-TBI) in ALL patients. We retrospectively analyzed 21 patients treated with HDACE-TBI, of whom 18 were in complete remission (CR) and three were in non-CR at transplantation. Although gastrointestinal toxicities were common, critical regimen-related toxicities were not seen in any patients. One patient demonstrated veno-occlusive disease, which could be controlled conservatively. The disease-free survival rate of 18 patients in CR at transplantation was 61%. These results demonstrate that the HDACE-TBI combination regimen is a feasible alternative to other preparatory regimens and does not increase the regimen-related toxicity.

Platelet-collagen interaction is important in primary hemostasis and collagen receptors on the platelet surface include membrane glycoprotein (GP) Ia/IIa and VI. Platelets from a 47-year-old woman with systemic lupus erythematosus (SLE) and a mild bleeding symptom showed a defective collagen-induced aggregation and an impaired adhesion to collagen surface. The patient's platelets had a markedly decreased content of GPVI. The patient had an antibody against GPVI in serum and the patient's plasma induced aggregation and release reaction of normal platelets. These findings indicate that GPVI is an important receptor for collagen on the platelet surface, and that anti-GPVI antibody activates the platelets, resulting in aggregation. This is the first documented case of SLE who acquired a platelet-aggregating anti-GPVI antibody.

Some cytokines are believed to play a role in the development of acute and chronic GVHD after allo-hematopoietic stem cell transplantation. It has been reported that TNF-α and IL-10 gene polymorphisms are associated with the production of those cytokines and the development of graft failure after organ transplantation and systemic lupus erythematosus. We examined whether TNF-α and IL-10 gene polymorphisms affect the severity of acute GVHD (aGVHD) and chronic GVHD (cGVHD). Sixty-two and 54 patients were available for the analysis of aGVHD and cGVHD, respectively. We analyzed the gene polymorphisms derived from pre- and post-transplant blood cells. Donor-derived TNF2 allele (A) was more frequently detected in patients with aGVHD III/IV than those aGVHD 0-II (2/6 vs 2/56) (P = 0.04). The donors of the patients with cGVHD more frequently possessed a greater number of alleles (allele 13 or more which contain 26 or more CA repeats) in IL-10.G than those without (13/26 vs 5/28) (P = 0.02), and the patients with cGVHD had more CA repeats in donor-derived IL-10.G than those without (mean = 25.2 vs 23.4) (P= 0.01). Donor-derived TNF-308 and IL-10.G alleles may contribute to severe aGVHD and cGVHD, respectively, and will help us distinguish those patients at high risk for GVHD. Bone Marrow Transplantation (2000) 26, 1317–1323.

Background Patients with acute promyelocytic leukemia (APL) patients present disseminated intravascular coagulation (DIC) that can result in life-threatening hemorrhagic complications. In 2008, the anticoagulant thrombomodulin alfa (TM-α, recombinant human soluble thrombomodulin) was approved for the treatment of DIC in Japan. After commercial launch, only small case series have been described in APL patients, and data from larger patients cohort are awaited. We accessed the clinical safety and beneficial effects of TM-α treatment in DIC patients with APL, by evaluating data on the open-label, multicenter, post-marketing surveillance study cohort. Patients and Methods A retrospective evaluation was carried out on a cohort of 172 patients with APL from the open-label, multicenter, post-marketing surveillance study of TM-α in Japan. Of all 172 patients, 31 were relapse/refractory APL patients and 141 were newly diagnosed. The newly diagnosed APL patients were generally received all-trans retinoic acid (ATRA) and anthracycline-containing induction therapy followed by consolidation therapy as previously reported by the Japan Adult Leukemia Study Group (Yanada et al, Eur. J. Haematol. 2007). The relapse/refractory APL patients were received arsenic trioxide, tamibarotene, and/or gemtuzumab ozogamicin. Early death (death within 30 days from the start of antileukemic treatment), severe hemorrhagic events, and the improvement of coagulopathy were analyzed. Results The study population consisted of 97 males and 75 females, with a median age of 58 (range 10 - 88). TM-α was administration by 380 ± 52.5 U/kg/day and the duration of dosing was 7.0 ± 6.3 days. In most patients (81 %), TM-α was started on or before the start of antileukemic treatment. Within the first 30 days, 12 patients (7 %) had severe hemorrhage and there were 24 (14 %) early deaths. Six of those (3.5% of 172 patients) were due to hemorrhage. TM-α treatment rapidly and significantly improved coagulopathy regardless of concomitant ATRA treatment (Table 1). Conclusions Although anticoagulation therapy during APL chemotherapy may induce severe hemorrhagic complication, our findings suggest that supportive care with TM-α ameliorates coagulopathy and reduces the risk of hemorrhagic early deaths in patients with APL. Disclosures: Watanabe: Asahi Kasei Pharma Corporation: Employment. Honda:Asahi Kasei Pharma Corporation: Employment.

Background and Purpose Imatinib therapy shows excellent efficacy for chronic myeloid leukemia (CML) patients. The five-year overall survival of chronic-phase CML patients treated with imatinib can be expected to reach over 90%. Now, the goal of therapy has become the achievement of therapy-free remission (TFR) for most patients. However, predictive markers for achieving deep molecular response (MR) or TFR are yet to be elucidated. The recently published European LeukemiaNet recommendations 2013, which are mostly based on Caucasian studies, show the importance of an early response at 3 months or 12 months after starting imatinib treatment to assess optimal response. Using the registry of our study group, we assessed whether early cytogenetic or molecular responses at 3 months and 12 months after starting imatinib are associated with the achievement of deep MR or long-term outcome in Japanese CML patients. Patients and Method We reviewed 135 CML patients in the registry of our study group. Imatinib was started between December 2001 and June 2008. We retrospectively analyzed 92 CML patients (35 patients with prior therapy before imatinib) who could be assessed for partial cytogenetic response (PCyR: Ph Results Seventy-six out of 92 patients (82.6%) achieved PCyR at 3 months. Forty out of 81 patients (49.3%) achieved MMR at 12 months. The patients with PCyR at 3 months showed significantly better overall survival (p=0.004) and higher cumulative achievement of deep MR (p=0.009) than the patients without PCyR. Overall survival between the patients with and without MMR at 12 months did not show a significant difference (p=0.06). However, the patients with MMR at 12 months showed significantly higher cumulative achievement of deep MR (p Conclusion Early cytogenetic and molecular responses at 3 months and 12 months after starting imatinib are also predictive factors for good prognosis and the achievement of deep MR in a registry of Japanese patients. Our data confirm the criteria of optimal response in European LeukemiaNet recommendations 2013 was appropriated for Japanese CML patients in practical setting. Disclosures: No relevant conflicts of interest to declare.

In order to assess the clinical implication of tissue factor pathway inhibitor (TFPI) in disseminated intravascular coagulation (DIC), plasma concentrations of TFPI were measured together with plasma tissue factor (TF) in 30 healthy subjects and 49 patients with DIC associated with a variety of underlying diseases. The mean TFPI concentration was elevated in patients with DIC at presentation (205.8 +/- SD 79.1 ng/ml) as compared with healthy subjects (97.3 +/- 22.2 ng/ml, P0.001). The mean plasma TF concentration in patients with DIC (412.7 +/- 445.7 pg/ml) was also higher than that in healthy subjects (137.5 +/- 50.6 pg/ml, P0.001). Elevated TF levels were found predominantly in patients with DIC caused by cancer and leukemia, whereas TFPI was elevated in all underlying disease categories. Plasma TFPI concentration did not correlate with plasma TF. In addition, hemostatic markers of DIC such as thrombin-antithrombin complex, prothrombin fragment 1 + 2, plasmin-plasmin inhibitor complex, FDP or fibrinogen did not correlate with TFPI. Serial determinations of plasma TFPI in each patient demonstrated that the behavior of TFPI was independent of the changes in plasma TF and other hemostatic parameters. These findings indicate that plasma TFPI does not decrease in DIC and is not valuable for monitoring the progress of DIC.

Recent progress in the measurements of the hemostatic markers enables us to assess the detailed profiles of hemostatic activation in various diseases. To evaluate the degree of hemostatic system activation in patients with cerebral thrombosis, detailed coagulation studies were performed in 28 patients with acute-phase cerebral thrombosis and in 36 with chronic-phase cerebral thrombosis, together with 6 with chronic-phase cerebral hemorrhage and 37 age-matched healthy volunteers. In both acute-phase and chronic-phase cerebral thrombosis, plasma levels of thrombin-antithrombin III complex, plasmin-alpha 2-plasmin inhibitor complex and D-dimer were significantly higher, and antithrombin III and protein C were significantly lower than those in the normal group. Plasma fibrinogen concentration was significantly higher in chronic-phase cerebral thrombosis than that in chronic-phase cerebral hemorrhage. No significant difference was found in these variables between acute-phase and chronic-phase cerebral thrombosis. In addition, there was no difference in these parameters between chronic phase cerebral hemorrhage and normal subjects. These findings indicate that a sustained activation of coagulation and fibrinolysis is present in cerebral thrombosis, and it might contribute to the pathogenesis of cerebral thrombosis.

Abstract 3751 Background and Purpose: The introduction of imatinib to the treatment regimen of chronic myeloid leukemia (CML) has markedly changed its outcome. Several reports have shown that the efficacy of imatinib correlates with the trough plasma concentration in CML patients. However, little is known about the correlation between plasma concentration and long-term outcomes, including adverse effects. We analyzed the correlation between long term tolerability and plasma concentration in Japanese patients. Patients: Forty-three patients with chronic phase CML who had good adherence with daily 300mg (n=8) or 400mg (n=35) imatinib administration at hospitals in our study group were enrolled in this study. The median age of patients starting imatinib was 57 years (range 15–82). Imatinib was started between Dec. 2001 and May 2007. Plasma trough concentration was examined on a day between Nov. 2007 and Aug. 2008. Median time from the start of imatinib until the plasma assay was 1548 days (range 91–2390). Results: Clinical data were reviewed in Sep 2011. We excluded patients who had been switched from imatinib to a second tyrosine kinase inhibitor (TKI) despite major molecular response (MMR). Median plasma concentration was 925 ng/ml (range 200–3280). Median follow-up was 2632 days (range 884–3496) and median follow-up after the plasma assay was 1216 days (range 361–1323). We divided the patients into two groups: the low plasma concentration group (925ng/ml n=21). Overall survival and achievement of complete molecular response (negative by RT-PCR) did not differ between the two groups. We defined all death, progression, switch to a second TKI and imatinib dose reduction as events. Also we defined death by adverse effect, imatinib dose reduction or switch to a second TKI due to adverse effects as intolerance-related events and progression, switch to a second TKI due to failure of achieve MMR or loss of MMR as efficacy-related events. Event-free survival (EFS) showed no difference between the two groups. However cumulative incidence of intolerance-related event was significantly higher in the high plasma concentration group than in the low plasma concentration group (whole observation period, p=0.02 by log-rank test; observation period after the plasma assay, p=0.01 by log-rank test). Grade 3 or 4 anemia occurred in 4 patients only in the high plasma concentration group. Cumulative incidence of efficacy-related event was slightly higher in the low plasma concentration group than the high plasma concentration group (whole observation period, p=0.06 by log-rank test; observation period after the plasma assay p=0.05 by log-rank test). Conclusion: Trough imatinib plasma concentration at steady-state after initiation of therapy has been demonstrated to have correlation with the early efficacy of imatinib. Our findings suggest that imatinib plasma concentration also correlates with intolerance or adverse effect in long-term clinical observation. Although further studies are required to confirm the current data prospectively, imatinib plasma level testing seems to be helpful to optimize clinical outcomes for patients with CML. Disclosures: No relevant conflicts of interest to declare.

To assess the degree of haemostatic system activation, plasma levels of prothrombin fragment 1 + 2 (F1 + 2), a direct indicator for thrombin generation in vivo, were measured in 49 patients with thrombotic disease undergoing long-term warfarin therapy (Thrombotest values < or = 40%). In these patients, vitamin K dependent coagulation factors (factors II, VII, IX and X) were decreased together with the anticoagulant proteins C and S, but the mean plasma concentration of F1 + 2 was significantly decreased compared with 48 healthy subjects. In warfarin-treated patients, F1 + 2 was positively correlated with the Thrombotest value, factors II, VII, IX and X. When analysed according to the intensity of anticoagulation, patients with Thrombotest values less than 30% showed a significant decrease in F1 + 2, but the mean F1 + 2 level was normal in patients with Thrombotests higher than 30%. These findings indicate that long-term oral anticoagulant therapy suppresses thrombin generation approximately in parallel to the decrease in coagulation factors, and levels of F1 + 2 lower than healthy subjects are observed when Thrombotest values are less than 30%.

Endothelial cell injury is thought to be one of the causative factors in thrombotic thrombocytopenic purpura (TTP). A novel index of endothelial injury, plasma thrombomodulin, was measured in 13 patients with acute TTP. The mean plasma concentration of thrombomodulin was elevated in patients with TTP (34.23 +/- 19.08 ng/ml) as compared with healthy subjects (16.99 +/- 2.63 ng/ml, P less than 0.001). Eight (61.5%) of 13 patients had high thrombomodulin values. Markedly elevated thrombomodulin levels were observed in TTP patients who had suffered from systemic lupus erythematosus, in whom plasma thrombomodulin was still elevated when they achieved remission. Five of these 13 patients with TTP had normal plasma levels of thrombomodulin. In addition, the plasma thrombomodulin concentrations were correlated well with von Willebrand factor antigen and tissue-type plasminogen activator antigen levels, both of which are released from stimulated or damaged endothelial cells. No difference was found in plasma thrombomodulin levels between patients who achieved remission and who did not. These findings suggest that the magnitude of the endothelial damage in TTP is variable among patients and that plasma thrombomodulin has limited clinical relevance to the severity of TTP.

Desmopressin acetate (DDAVP) is known to stimulate the release of tissue-type plasminogen activator (t-PA) from endothelial cells, but it is unclear whether the increased t-PA actually elicits the plasmin generation and fibrin(ogen)olysis in the circulating blood. We measured plasma levels of plasmin-alpha 2-plasmin inhibitor complex, fibrinogen degradation products (FgDP) and fibrin degradation products (FbDP) following desmopressin infusion in 19 patients with bleeding disorders or thrombophilia. Administration of desmopressin (0.3-0.4 microgram/kg) produced a 4.0-fold increase in plasmin-alpha 2-plasmin inhibitor complex at 30 min, whereas neither FgDP nor FbDP was elevated significantly. These findings indicate that desmopressin infusion provokes the generation of plasmin in vivo, but most of the plasmin generated is complexed to alpha 2-plasmin inhibitor and does not degradate fibrin or fibrinogen.

Patients with liver disease frequently have hemostatic abnormalities which include accelerated fibrinolysis. In order to assess the fibrinolytic state in liver disease, plasma levels of fibrinogenolysis products (FgDP), fibrinolysis products (FbDP), and fibrinogenolysis plus fibrinolysis products (TDP) were measured with newly developed enzyme-linked immunosorbent assays based on monoclonal antibodies in 36 patients with liver disease (six patients with acute hepatitis, seven with chronic hepatitis, ten with liver cirrhosis, 11 with hepatocellular carcinoma, and two with intrahepatic cholestasis). As compared with healthy subjects, mean plasma levels of FbDP (1,083 +/- SD 1,254 vs. 236 +/- 100 ng/ml, P = 0.005) and TDP (1,773 +/- 1,814 vs. 669 +/- 212 ng/ml, P = 0.001) were significantly elevated in patients with liver disease, whereas FgDP was normal (389 +/- 202 vs. 396 +/- 132 ng/ml, P = 0.87). Plasma FbDP correlated very well with TDP (r = 0.986, P less than 0.00001) in liver disease. In addition, FbDP and TDP but not FgDP correlated with plasma concentrations of thrombin-antithrombin III complex. When plotted by the disease categories, the magnitude of elevations of FbDP and TDP was the most prominent in acute hepatitis followed by hepatocellular carcinoma. These findings indicate that activation of fibrinolysis occurs following thrombin generation, but increased primary fibrinogenolysis is rare in liver disease.

SummaryIn order to assess precisely the fibrinolytic state in disseminated intravascular coagulation (DIC), plasma levels of fibrinogenolysis products (FgDP), fibrinolysis products (FbDP) and fibrinogenolysis plus fibrinolysis products (TDP) were measured with newly developed enzyme-linked immunosorbent assays based on monoclonal antibodies in 72 patients with DIC at presentation. Not only FbDP and TDP but also FgDP were markedly elevated in patients with DIC. When analyzed according to the underlying disease categories, the relative proportion of FgDP to TDP was high in patients with acute promyelocytic leukemia and vascular diseases, and it was the lowest in patients with sepsis. Correlation analysis revealed that plasma levels of FgDP correlated negatively with alpha 2-antiplasmin and positively with plasmin-alpha 2-antiplasmin complex (PAP) and a ratio of PAP to thrombin-antithrombin III complex (TAT). These findings indicate that besides fibrinolysis, fibrinogenolysis is markedly accelerated in the majority of the patients with DIC.

In order to assess the thrombin and plasmin generation in vivo in disseminated intravascular coagulation (DIC), plasma levels of thrombin-antithrombin III (ATIII) complex (TAT) and plasmin-alpha 2-antiplasmin (a2AP) complex (PAP) were measured together with standard coagulation and fibrinolytic parameters in 80 patients with DIC. Both TAT and PAP were markedly elevated in patients with DIC. When plotted by the underlying disease categories, differences in the magnitude of the elevations of these complexes were recognized among groups. Patients with acute promyelocytic leukemia (APL) had the highest PAP, the lowest TAT/PAP ratio, low a2AP, and low fibrinogen, indicating that the most excessive fibrinolysis can occur in APL. Similar profiles, although less marked, were observed in patients with other leukemias and vascular diseases. Patients with sepsis showed the highest TAT/PAP ratio and the lowest PAP with no decrease in a2AP or fibrinogen, demonstrating a relatively impaired fibrinolysis. Patients with cancer had a relatively high TAT and high TAT/PAP ratio. In addition, both TAT and PAP were markedly elevated in patients with shock. From these, it was suggested that, although laboratory manifestations in DIC are extremely variable from patient to patient, underlying disorders are, at least in part, responsible for the observed variations. Recognition of this variable activation of coagulation and fibrinolysis would be helpful for the proper management of patients with DIC.

We discussed utility of cell cycle and phenotypic analysis of acute promyelocytic leukemia (APL) cells using 7AAD/PY for the prediction of efficacy and risks of all-trans retinoic acid (ATRA) and granulocyte colony-stimulating factor (G-CSF) administration to patients with APL. Serial changes in phenotype and cell kinetics of APL cells from two patients were analyzed during ATRA administration. CD15 and CD11b were expressed on the APL cells in vivo as neutrophil maturation markers, while growth activity of the cells was decreased during ATRA administration. Using 7AAD/PY, changes in phenotype and cell kinetics were clearly detected after 2 days of cultivation with ATRA and/or G-CSF. In one patient, APL cells harvested from marrow during the first 3 weeks of ATRA administration showed distinct growth sensitivity to G-CSF ex vivo, and the cells harvested after a 4-week exposure to ATRA appeared to have lost this sensitivity. In this patient, G-CSF could be safely administered after 4 weeks of ATRA therapy. 7AAD/PY analysis is useful for predicting growth sensitivity of APL cells to G-CSF during ATRA administration.

SummaryIt has been shown that platelets from patients with platelet- type von Willebrand’s disease (vWD) agglutinate upon the addition of human von Willebrand factor (vWF) in the absence of ristocetin or botrocetin, suggesting that platelet membrane receptors for human vWF is abnormal. The present work reports the platelet agglutinability on stimulation with bovine vWF in platelet-type vWD. Platelets in patient platelet-rich plasma or washed platelet suspensions and patient platelets treated with formalin agglutinated in the presence of markedly lower concentrations of bovine vWF than those required for normal platelets. This finding provides additional evidence that platelet-type vWD platelets have abnormal expression of binding sites for vWF on their surface, and supports that platelet receptors for bovine vWF are identical or very close to those for human vWF.

Serum of a 67-year-old male patient with smoldering multiple myeloma was shown to contain two monoclonal immunoglobulins, IgG and IgA. For the initial seven months, monoclonal IgG was predominantly elevated. During the next one year and eight months, however, serum concentration of the monoclonal IgA increased, with a concomitant decrease of IgG. N- terminal amino acid sequences of heavy and light chains separated from monoclonal IgG and IgA were analyzed. Both light chains were lambda- type and showed identical amino acid sequences of variable regions. The heavy chains also had the same N-terminal amino acid sequence between IgG and IgA. These results strongly suggest that two monoclonal proteins, IgG and IgA, in this patient were produced by B lymphocytes within a clone and that class switch from IgG to IgA in immunoglobulin production during B cell differentiation has taken place in the clinical course of this case.

Abnormal factor IX was isolated from the plasma of a patient with hemophilia B Kashihara and two patients with hemophilia BM. The F.IX was purified to homogeneity by using monoclonal anti-F.IX-Sephrose, heparin-Sepharose and DEAE-Sephadex A-50 affinity chromatography successively. The isolated proteins have the same molecular weight and the same mobility on crossed immunoelectrophoresis as normal F.IX. The limited proteolysis of purified proteins was induced by F.XIa/Ca 2+ or by RVV-X/ Ca 2+ . A time course study showed that F.IX Nagoya seemed to be cleaved by neither F.XIa nor RVV-X, F.IX Kashihara was cleaved partially by F.XIa but not by RVV-X, and that F.IX Niigata was cleaved completely at the rate similar to normal F.IX, though the resultant product of F.IX Niigata did not show any F.IXa activity. These results favored the view that hemophilia B or BM is a heterogeneous disorder.

The profile of blood coagulation and fibrinolysis was studied in detail in eight patients with acute thrombotic thrombocytopenic purpura (TTP). In the majority of the patients, fibrinogen, factor XIII, antithrombin III, alpha 2-plasmin inhibitor, plasminogen, and alpha 2-macroglobulin were normal, whereas FDP, plasmin-alpha 2-plasmin inhibitor complex, and tissue-type plasminogen activator antigen were marginally or moderately elevated. Low fibronectin values were observed in four patients. Protein C and C4b-binding protein were nearly normal, whereas total protein S and free protein S were reduced in five and six patients, respectively. A positive correlation was found between total protein S and C4 and between free protein S and C3. von Willebrand factor antigen (vWf:Ag) and ristocetin cofactor (RCof) were either normal or elevated, but RCof/vWf:Ag ratio was decreased in seven patients. Crossed immunoelectrophoresis and sodium dodecyl sulfate (SDS)-agarose gel electrophoresis revealed that the large vWf multimers were either absent from or relatively decreased in all patients except one. In addition, one patient had unusually large vWf multimers, and a low-molecular-weight vWf fragment was apparently observed in three patients. These findings indicate that the intravascular generation of thrombin and plasmin was minimal in TTP and suggest that the alterations of the vWf molecule were caused not only by consumption through its participation in platelet thrombus formation but also by accelerated proteolysis. Low protein S values would be related to the immunological abnormalities underlying TTP.

We studied four patients who showed aggregation of platelets in platelet-rich plasma at lower concentrations of ristocetin than those required for normal platelet-rich plasma and who demonstrated an increased capacity of the platelets to bind normal von Willebrand factor. The four patients were from two Japanese families. Platelets from one family aggregated spontaneously in vitro, and platelets from both families aggregated upon the addition of normal plasma and cryoprecipitate, in the absence of ristocetin or other agonists. Analysis of the multimeric composition of von Willebrand factor by sodium dodecyl sulfate-agarose gel electrophoresis revealed a decrease in large multimers or a decrease in both large and intermediate multimers in plasma, but normal multimers in platelets. 1-Deamino-[8-D- arginine]-vasopressin caused by an immediate appearance of larger multimers in plasma, followed by the rapid disappearance of these multimers from circulating plasma. Analysis of platelet membrane glycoproteins from the patients showed that there were two distinct bands in the glycoprotein I region; one migrated in a slower region and the other in a faster region than normal glycoprotein Ib. We suggest that the platelet receptor abnormality in these patients is related to this abnormality of glycoprotein Ib.

Blood platelets from patients with platelet-type von Willebrand disease (vWD) aggregate upon the addition of human von Willebrand factor (vWf) in the absence of ristocetin or other stimulating factors. We measured quantitatively the ability of vWf to induce directly aggregation of platelet-type vWD platelets (platelet-type vWD platelet aggregating factor [PT-PAF]). Cryoprecipitate and factor VIII concentrates were used as a source of vWf of various multimeric composition. The PT-PAF activity was dependent on the multimer size of vWf, like in the case of ristocetin cofactor (RCof) activity. However, PT-PAF activity was not equivalent to RCof activity and the relative PT-PAF/RCof ratio ranged from 1.00 to 0.18 in the materials studied. The preparations containing the higher-molecular-weight multimers had higher PT-PAF/RCof ratio. These findings suggest that PT-PAF activity is a functional expression of more highly polymerized multimers of vWf as compared with RCof activity. Measurement of PT-PAF would serve as a novel functional assay of vWf.