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Prothrombinase activities of platelets have been measured in diluted platelet-rich plasma using a chromogenic substrate assay and purified coagulation factors. No abnormalities in prothrombinase activities were found for platelets from patients with storage pool disease (dense-body deficiency), grey platelet syndrome, and Glanzmann's thrombasthenia. It is concluded that neither release of dense bodies and alpha-granules nor aggregation of platelets are essential prerequisites for exposure of a procoagulant surface. Platelets from patients with Bernard-Soulier syndrome, however, have approximately 10-fold higher prothrombinase activities in the non-stimulated form than normal non-stimulated platelets. The increased procoagulant activity cannot be completely ascribed to an increase in platelet size. It is suggested that the increased prothrombinase activity reflects an increased exposure of phosphatidylserine at the outer surface of non-stimulated Bernard-Soulier platelets, earlier described by Perret et al (1983).

Summary. Background: Fondaparinux is a synthetic pentasaccharide, which selectively inhibits coagulation factor (F) Xa, and is registered for prevention of venous thromboembolism following hip fracture, hip replacement, and knee replacement surgery. Recently, it was shown that recombinant FVIIa (rFVIIa) reverses anticoagulant effects of fondaparinux in healthy volunteers. Objectives: In this study, we have explored the in vitro and ex vivo effects of rFVIIa on clot formation and thrombin-activatable fibrinolysis inhibitor (TAFI)-mediated down-regulation of fibrinolysis after fondaparinux administration. Methods: In vitro clot lysis assays were performed in pooled normal plasma from healthy volunteers to which fondaparinux was added, and in serial samples from healthy volunteers who received a single bolus dose of fondaparinux, a single bolus dose of rFVIIa, or both. Results and conclusions: Fondaparinux significantly delayed clot formation, and clot lysis was significantly increased due to decreased activation of TAFI. Addition of recombinant FVIIa corrected the inhibited clot formation induced by fondaparinux, and the acceleration of clot lysis was partially reversed. In vivo administration of fondaparinux (10 mg) to healthy volunteers similarly resulted in accelerated plasma clot lysis. Subsequent administration of rFVIIa (90 µg kg−1) normalized the clot lysis time up to 6 h postadministration. rFVIIa might be a good therapeutic option in patients treated with fondaparinux who develop bleeding complications, since both clot formation as well as fibrinolytic resistance are improved.

Summary. d-Dimer measurement is a promising tool in the exclusion of venous thrombosis. New d-dimer assays have been introduced, but need clinical validation. Our objective was to evaluate the clinical usefulness of four relatively new d-dimer assays and a classical ELISA in outpatients suspected for deep venous thrombosis. In 537 patients, participants in a large prospective management study using a clinical probability score and a d-dimer measurement (Tina-quant®), additional samples were taken for d-dimer measurement using the Asserachrom ELISA®, the VIDAS New®, the STA-LIA® and the Miniquant® assay. Performances of each test were calculated using clinical data during a 3-month follow-up. Thrombosis was detected in 224 patients (42%). The area under the ROC curve was significantly higher for the Tina-quant as compared to the other assays. Using standard cut-off values, sensitivity, negative predictive value (NPV) and specificity of the Asserachrom were 97, 94 and 33%, respectively. For the VIDAS New, values were 100, 96 and 8%, respectively. The Tina-quant showed values of 99, 98 and 41%, respectively, and the STA-LIA 98, 95 and 32%. Values for the Miniquant were 95, 94 and 52%. The d-dimer assays in our study all show a high sensitivity and negative predictive value, but none of the assays reached an NPV of > 98% at standard cut-off values. d-Dimer assays with a low specificity still necessitate additional diagnostic tests in the majority of the patients.

The development of arthropathy is a serious complication of severe haemophilia. With the use of prophylaxis, bleeds can be prevented and arthropathy delayed. We investigated whether an individually tailored prophylactic regimen can prevent arthropathy and whether it had a similar effect on orthopaedic outcome compared with that of a high-dose regimen. Efficacy was determined clinically and by radiographs of six major joints. Prophylaxis was started in 70 patients at a mean age of 4.1 years. Mean follow-up was 15.6 years (range 8-24.5 years). The mean factor VIII consumption was 2319 IU/kg/year. The mean number of joint bleeds was 3.5/year and the mean clinical score (maximum score 90) was 1.0, with a mean Pettersson joint score (maximum score 78) of 3.0 at a mean age of 13.5 years. In conclusion, long-term, early-onset, individualized prophylaxis in haemophilia is feasible and prevents arthropathy.

Factor VII (FVII) deficiency is a rare haemorrhagic condition, normally inherited as an autosomal recessive trait, in which clinical presentation is highly variable and correlates poorly with laboratory phenotype. The FVII (F7) gene was sequenced in 48 unrelated individuals with FVII deficiency, yielding a total of 23 novel lesions including 15 missense mutations, 2 micro-deletions, 5 splice junction mutations and a single base-pair substitution in the 5' untranslated region. Family studies were performed in order to distinguish the contributions of individual mutant F7 alleles to the clinical and laboratory phenotypes. Specific missense mutations were evaluated by molecular modelling in the context of the FVIIa-tissue factor crystal structure. Single base-pair substitutions in splice sites and the 5' untranslated region were studied by in vitro splicing assay and luciferase reporter gene assay, respectively. All probands were also typed for four previously reported F7 polymorphisms. In the majority of cases of FVII deficiency studied here, consideration of both mutational and polymorphism data permitted the derivation of plausible explanations for the FVII activity and antigen levels measured in the laboratory. Inter-familial variation in FVII activity and the antigen levels of heterozygous relatives of probands was found to be significantly higher than intra-familial variation, consistent with the view that the nature of the F7 gene lesion(s) segregating in a given family is a prime determinant of laboratory phenotype. Although no relationship could be discerned between laboratory phenotype and polymorphism genotype, the frequencies of the A2 and M2 polymorphic alleles were significantly higher in the FVII-deficient individuals tested than in controls. This suggests that the presence of these alleles may have served to increase the likelihood of pathological F7 gene lesions coming to clinical attention.

We evaluated the efficacy and toxicity of different doses of donor T cells given with donor leukocyte infusions (DLI) as treatment for relapse of various hematologic malignancies after allogeneic bone marrow transplantation (BMT). We also studied whether DLI treatment was more effective if circulating T cells were exclusively of donor origin (complete donor T cell chimeras) as compared with T cells originating from both donor and recipient (mixed T cell chimeras). Twenty-eight patients were studied of whom 24 had a complete donor T cell chimerism. The malignancies were as follows: chronic myeloid leukemia (CML) in chronic phase (CP) (n = 9); more advanced CML (n = 5); multiple myeloma (MM) (n = 5); acute leukemia (AL) (n = 9). T cell doses varied from 0.1 x 10(7) to 33 x 10(7) T cells/kg. Eight patients received two to four DLI courses because they failed to respond to one course. Thirteen of 14 patients with CML, including four patients with more advanced CML, achieved complete remission (CR). All five patients with MM responded, including three CRs. Six patients (three with CML, three with MM) responded only after two to four DLI courses. Patients with CML-CP were likely to respond to as few as 1 x 10(7) T cells/kg whereas patients with MM generally responded when they receivedor = 10 x 10(7) T cells/kg. However, despite the infusion of high T cell doses (up to 32 x 10(7) T cells/kg), practically all patients with AL failed to respond. The likelihood of response was strongly related to the occurrence of graft-versus-host disease (GVHD) in patients with CML and MM (P = 0.0002), although GVHD was not helpful for patients with AL. Higher T cell doses (or = 10 x 10(7)/kg) induced serious GVHD (n = 17) and marrow aplasia (n = 5), and GVHD was directly or indirectly the cause of death for six patients. Finally, there were no obvious differences in responses between complete donor T cell chimeras and mixed T cell chimeras.

Objective This study was designed to estimate the predictive value of the first neonatal platelet count for the second neonate in women with immune thrombocytopenic purpura (ITP). Methods Data of 34 patients, repeatedly pregnant while they had ITP, were prospectively collected in two study centers between 1984 and 1995. The main outcome measure was neonatal thrombocytopenia. Results Early neonatal platelet counts (ie, umbilical cord count or count during the first 24 hours of life) between siblings were correlated (r = .73; 95% confidence interval (CI) for the correlation coefficient 0.52, 0.86). Severe thrombocytopenia (less than 50 × 109/L) at birth did not occur in any of the 27 siblings of infants with birth platelet levels above 50 × 109/L. Also the second sibling's nadir neonatal platelet counts during the first 2 weeks of life were correlated with those of the first sibling (r = .76; 95% CI for the correlation coefficient 0.58, 0.88). In those cases in which the first sibling had a lowest platelet count above 100 × 109/L (n = 19), the second sibling never became thrombocytopenic. Conclusion The platelet count of the first sibling can be used to counsel women with ITP, and may be helpful in their management.

A 21-year-old Surinam boy suffering from a combined heterozygous sickle-cell beta-thalassaemia presented with an extreme anaemia (haemoglobin concentration 0.7 mmol/l) and severe haemolysis without signs of increased erythropoiesis. An acute B19 parvovirus infection was diagnosed by a transient increase of IgM anti-B19 parvovirus titres and a positive polymerase chain reaction for B19 parvovirus. The patient recovered spontaneously after a short period of supportive care with red cell transfusions and mechanical ventilation.

SummaryP-selectin is a 140 kD protein found in the α-granules of platelets and the Weibel-Palade bodies of endothelial cells. On cell activation it is expressed on the cell surface and also secreted into plasma. Whether the circulating soluble P-selectin (sP-selectin) originates from platelets, endothelial cells, or both, is not known. We studied the level of sP-selectin in diseases with different platelet counts, with or without evidence of endothelial cell activation. Endothelial cell activation was confirmed by the detection of sE-selectin and EDl-fibronectin. A significant positive correlation between platelet count and sP-selectin concentration was observed in healthy controls, and in patients with thrombocytopenia due to bone marrow aplasia, or with thrombocytosis (r = 0.85; n = 47; p This study suggests that platelets are the major source of circulating sP-selectin in healthy individuals. Endothelial cell activation is associated with an increased sP-selectin concentration per platelet.

Collagen type IV is a sheet-forming collagen and a major constituent of the vessel wall. To find out which conditions are important for platelet adhesion to collagen type IV, we performed perfusion studies with anticoagulated blood in parallel plate perfusion chambers. The role of divalent cations was investigated by using plasmas with variable concentrations of Mg2+ and Ca2+ions. When Mg2+ concentration was decreased from 2.00 mmol/L to 0.25 mmol/L at a fixed Ca2+ concentration of 1.25 mmol/L, platelet coverage on the collagen type IV surface decreased from 22.8% +/-1.8% (n = 4) to 4.6% +/- 0.6% (n = 4) at a shear rate of 1,600 s-1. Also, platelet aggregate formation on collagen type IV was strongly impaired. A monoclonal antibody against the glycoprotein (Gp) Ib receptor and von Willebrand factor (vWF)-depleted plasma reduced the platelet coverage to collagen type IV to, respectively, 10% and 45% of the control value. Electron microscopy showed that vWF was only present between platelets and between the platelet and the collagen type IV surface, but did not bind elsewhere to collagen type IV. These data indicate that collagen type IV is a reactive collagen for platelets. Differences in physiologic plasma magnesium concentrations may in part explain the differences in platelet reactivity to collagen type IV between individuals, and perhaps contribute to differences in the risk for thrombosis.

SummaryPlatelet concentrates stored at room temperature deteriorate. The so-called storage lesion is characterised by morphological changes and a loss of functionality. To find an assay for early platelet activation in platelet concentrates the morphological score, β-TG release and P-selectin expression were determined, and compared with the amount of soluble P-selectin. An ELISA was used to quantify soluble P-selectin in the storage medium. We found a significant correlation between the amount of soluble P-selectin and the percentage of P-selectin positive platelets (flow-cytometric analysis) (r = 0.7449; p

P-selectin is an alpha granule membrane associated glycoprotein in platelets (P1) expressed on the surface following exposure to secretagogues in suspension. It is not clear, whether P-selectin is transported from granule membranes to the P1 surface, or released by surface-activation (SfA). In the present study washed P1 were allowed to interact with grids for different periods of time (5-20 min), fixed briefly and exposed to a monoclonal antibody to P-selectin. Grids were washed and exposed to goat anti-mouse IgG antibody coupled to 10 nm gold particles. Examination in the electron microscope revealed a differential distribution of the gold probe on SfA P1. Discoid P1 did not express P-selectin. Early dendritic P1 revealed a few gold probes for P-selectin near the central zone. Late dendritic P1 expressed P-selectin on P1 bodies and some on pseudopods. On fully spread P1 P-selectin probes were evenly distributed, but more concentrated on the peripheral margin than the central zone. Results demonstrate that P-selectin is released from SfA P1. Its initial expression in the central zone suggests it reaches the surface through channels of the open canalicular system. The centrifugal movement of P-selectin is opposite in direction to translocation of mobile receptor-ligand complexes.

SummaryThromboembolic complications are frequently observed in patients with systemic lupus erythematosus (SLE). Significant associations have been reported between these complications and the presence of antiphospholipid antibodies, notably the lupus anticoagulant and anti-cardiolipin antibodies. Factor V Leiden is a genetic disorder associated with an increased risk of venous thrombosis. We studied these factors in 173 patients with SLE in relation to both arterial and venous thrombosis. The frequency of factor V Leiden in SLE patients is comparable to that in the Dutch population (5%) and a risk factor for venous thrombosis (odds ratio 4.9; Cl 1.2-19.6), but not for arterial thrombosis. The lupus anticoagulant is a risk factor for both arterial thrombosis (odds ratio 7.1; Cl 2.9-17.4) and venous thrombosis (odds ratio 6.4; Cl 2.7-15.4). From multivariate analysis, both the lupus anticoagulant and factor V Leiden appeared independent risk factors for venous thrombosis.

In patients with hemophilia A and inhibitory alloantibodies against factor VIII, various dosage schedules are used to obtain immune tolerance. In this study, we have evaluated the results of 13 years of low-dose immune tolerance induction and factors that are predictive of a positive result. The effect of immune tolerance induction in relation to age at inhibitor development, number of exposure days, age at start of therapy, maximum inhibitor titer, factor VIII products involved, and virologic status were determined. We evaluated 24 patients with severe hemophilia A and inhibitors who were treated with regular infusions with low-dose (25 U/kg every other day) factor VIII to obtain immune tolerance. In 21 of 24 patients (87%), immune tolerance induction was successful. The response time was determined by two factors: the highest inhibitor level and the age at inhibitor development. In patients with maximum inhibitor levels of less than 40 Bethesda units (BU)/mL, immune tolerance was obtained sooner than in patients with inhibitor levels exceeding 40 BU/mL (P = .005). Patients in whom an inhibitor developed before the age of 2.5 years also tended to have a quick immune response (P = .014). Immune tolerance with low-dose factor VIII is often successful in hemophilia A patients with inhibitors. Young children and patients with maximum inhibitors of less than 40 BU/mL show a relatively rapid response.

In patients with hemophilia A and inhibitory alloantibodies against factor VIII, various dosage schedules are used to obtain immune tolerance. In this study, we have evaluated the results of 13 years of low-dose immune tolerance induction and factors that are predictive of a positive result. The effect of immune tolerance induction in relation to age at inhibitor development, number of exposure days, age at start of therapy, maximum inhibitor titer, factor VIII products involved, and virologic status were determined. We evaluated 24 patients with severe hemophilia A and inhibitors who were treated with regular infusions with low-dose (25 U/kg every other day) factor VIII to obtain immune tolerance. In 21 of 24 patients (87%), immune tolerance induction was successful. The response time was determined by two factors: the highest inhibitor level and the age at inhibitor development. In patients with maximum inhibitor levels of less than 40 Bethesda units (BU)/mL, immune tolerance was obtained sooner than in patients with inhibitor levels exceeding 40 BU/mL (P = .005). Patients in whom an inhibitor developed before the age of 2.5 years also tended to have a quick immune response (P = .014). Immune tolerance with low-dose factor VIII is often successful in hemophilia A patients with inhibitors. Young children and patients with maximum inhibitors of less than 40 BU/mL show a relatively rapid response.

Elucidation of a novel pathogenomic mechanism using genome-wide long mate-pair sequencing of a congenital t(16;21) in a series of three RUNX1 -mutated FPD/AML pedigrees

Platelet membrane glycoprotein IV (GPIV) is a cell-surface glycoprotein that has been proposed as a receptor for collagen. Recently, it has been shown that platelets with the Naka-negative phenotype lack GPIV on their surface, whereas donors with this phenotype are healthy and do not suffer from hematologic disorders. In this study, we compared Naka- negative platelets with normal platelets in adhesion to collagen types I, III, IV, and V and the extracellular matrix of endothelial cells (ECM) under static and flow conditions. No differences in platelet adhesion and subsequent aggregate formation on the collagens types I, III, and IV were observed under static and flow conditions. Adhesion of both homozygous and heterozygous Naka-negative platelets to collagen type V was strongly reduced under static conditions. Collagen type V was not adhesive under flow conditions. No difference in platelet adhesion to ECM was observed, which suggests that GPIV is not important in adhesion to subendothelium, for which ECM may serve as a model. These results indicate that GPIV is not a functional receptor for collagen under flow conditions.

Despite prophylaxis with immunosuppressive drugs, severe acute graft- versus-host disease (GVHD) remains a major cause of morbidity and mortality in patients transplanted with unmodified bone marrow (BM) grafts from HLA-identical siblings. Although T-cell depletion of the BM graft has evolved as the most effective method to prevent severe acute GVHD, this beneficial effect is counterbalanced by an increased rate of graft failure and relapse of the disease. To find an approach to T-cell depletion that may avoid these extreme risks, we gave BM recipients a fixed low number of 1 x 10(5) donor T cells per kilogram of recipient's body weight in the graft. This corresponds with 99% T-cell depletion and is achieved by the addition of T cells to the graft that was previously depleted of T cells. A total of 70 patients with hematologic malignancies or aplastic anemia, including 40 patients with standard- risk leukemias, received BM grafts, depleted of T cells according to this approach, from HLA-identical siblings. The preparative regimen consisted of cyclophosphamide and total body irradiation. The patients also received a short course of cyclosporine posttransplant. Graft failure did not occur. Acute GVHD, only grade I or II, was seen in 70% of the patients and was limited to the skin in all patients. Chronic GVHD occurred in 31% of the patients and, with the exception of 1 patient, was limited to the skin as well. Relapse occurred in 3 of 40 (8%) patients with standard-risk leukemias, resulting in a projected survival at 5 years of 80%. Patients with standard-risk diseases had a procedure-related mortality of 11%. Quality of life, determined 1 year after BM transplant, was good in almost all patients with standard-risk diseases. Thus, this approach of T-cell depletion may be an approach that avoids the development of severe acute and chronic GVHD without damaging the function or antileukemic effect of the graft and that has a low transplant-related morbidity and mortality.

Platelet adhesion to fibrillar collagens (types I, II, III, and V) and nonfibrillar collagens (types IV, VI, VII, and VIII) was investigated in the presence of physiologic concentrations of divalent cations under conditions of stasis and flow. Under static conditions, platelet adhesion was observed to collagen types I through VII but not to type VIII. Under flow conditions, platelet adhesion to collagen types I, II, III, and IV was almost independent of shear rates above 300/s. Collagen type V was nonadhesive. Platelet adhesion to collagen type VI was shear rate-dependent and optimal at a rate of 300/s. Collagen types VII and VIII showed minor reactivity and supported platelet adhesion only between shear rates 100 to 1,000/s. Monoclonal antibody (MoAb) 176D7, directed against platelet membrane glycoprotein Ia (GPIa; very late antigen [VLA]-alpha 2 subunit), completely inhibited platelet adhesion to all collagens tested, under conditions of both stasis and flow. Platelet adhesion to collagen type III at shear rate 1,600/s was only inhibited for 85%. The concentration of antibody required for complete inhibition of platelet adhesion was dependent on the shear rate and the reactivity of the collagen. An MoAb directed against GPIIa (VLA-beta subunit) partially inhibited platelet adhesion to collagen. These results show that GPIa-IIa is a major and universal platelet receptor for eight unique types of collagen.

PURPOSE To assess whether the presence of enhanced multiple drug resistance (MDR)-1 gene expression in multiple myeloma (MM) patients predicts survival, as well as response to vincristine, doxorubicin, and dexamethasone (VAD) chemotherapy. PATIENTS AND METHODS Sixty-three MM patients refractory to alkylating therapy were studied. The presence of the MDR-1 gene product, a 170-kd glycoprotein (P-170), was analyzed in bone marrow plasma cells by means of the alkaline phosphatase (APAAP) technique using the P-170-specific monoclonal antibody (MoAb) C219. The prognostic value of MDR-1 gene expression, examined before VAD treatment, was compared with other established prognostic factors including beta 2-microglobulin, albumin, lactate dehydrogenase (LDH), and the plasma cell labeling index. RESULTS Fifty-nine percent of all samples were P-170-positive. No association could be demonstrated between response to VAD and MDR-1 gene expression (chi 2 P = .359), in contrast to high serum beta 2-microglobulin levels, which were positively correlated with response (P = .006). P-170-positive and -negative patients showed a median survival duration of 23 and 22 months, respectively, a difference that was not statistically significant (P = .9). beta 2-microglobulin, LDH, albumin, and the plasma cell labeling index were all significantly correlated with survival. CONCLUSION These results indicate that other mechanisms of resistance must be involved in MM apart from MDR. The role of MDR status at this stage of disease may be biased by the major contribution of dexamethasone to induction of response by VAD in MM patients.

Department of Hematology, University Hospital, Leiden, The NetherlandsSummaryAfter the mtroduction of a new pasteunsed factor VIII concentrate(Factor VIIICPS-P) m The Netherlands rn June 1990, an mcrease m theoccurrence of inhibitois m hemophihaA patients was reported Theclinical course of this gtoup of Inhibitors (n = 12) was compared withhemophilia patients in whom an Inhibitor developed before June 1990(classic Inhibitors) (n = 32) Stnkmg differences were found betweenboth groups not only m patient age (median 22 yeais versus 8 yeais)and number of exposure days ( 1000 versus

The aim of this investigation was to identify domains of collagen type I that can support platelet adhesion under flow conditions. Four cyanogen bromide (CB) fragments composing 87% of the collagen alpha 1(I)-chain were studied under static and flow conditions. Under static conditions, bovine and human collagen fragment alpha 1(I)CB3 induced aggregate formation, whereas alpha 1(I)CB7 and alpha 1(I)CB8 supported adhesion of dendritic and contact platelets. Bovine alpha 1(I)CB6 weakly supported platelet adhesion. At shear rate 300/s, collagen fragment alpha 1(I)CB3 strongly supported platelet adhesion, whereas lower platelet adhesion was observed to alpha 1(I)CB7 and alpha 1(I)CB8. The fragment alpha 1(I)CB6 did not support platelet adhesion under flow conditions. Adhesion to alpha 1(I)CB3 was completely inhibited by a low concentration (0.6 IgG microgram/mL) of anti-GPIa monoclonal antibody (MoAb), whereas this concentration of antibody partially inhibited adhesion to alpha 1(I)CB7 and alpha 1(I)CB8. At higher concentrations (3 micrograms/mL) the anti-glycoprotein Ia (GPIa) antibody completely inhibited adhesion to alpha 1(I)CB8 and further reduced adhesion to alpha 1(I)CB7. Platelet adhesion to alpha 1(I)CB3, alpha 1(I)CB7, and alpha 1(I)CB8 was strongly inhibited by an anti-GPIb MoAb. A MoAb against the GPIb-binding site of von Willebrand factor (vWF) strongly inhibited platelet adhesion to alpha 1(I)CB7 and alpha 1(I)CB8, whereas platelet adhesion to alpha 1(I)CB3 was not inhibited. We conclude that under flow conditions alpha 1(I)CB3, alpha 1(I)CB7, and alpha 1(I)CB8 support GPIa/IIa-dependent platelet adhesion. The GPIb-vWF interaction is important under flow conditions for adhesion to alpha 1(I)CB7 and alpha 1(I)CB8 and probably also to alpha 1(I)CB3.

The aim of this investigation was to identify domains of collagen type I that can support platelet adhesion under flow conditions. Four cyanogen bromide (CB) fragments composing 87% of the collagen alpha 1(I)-chain were studied under static and flow conditions. Under static conditions, bovine and human collagen fragment alpha 1(I)CB3 induced aggregate formation, whereas alpha 1(I)CB7 and alpha 1(I)CB8 supported adhesion of dendritic and contact platelets. Bovine alpha 1(I)CB6 weakly supported platelet adhesion. At shear rate 300/s, collagen fragment alpha 1(I)CB3 strongly supported platelet adhesion, whereas lower platelet adhesion was observed to alpha 1(I)CB7 and alpha 1(I)CB8. The fragment alpha 1(I)CB6 did not support platelet adhesion under flow conditions. Adhesion to alpha 1(I)CB3 was completely inhibited by a low concentration (0.6 IgG microgram/mL) of anti-GPIa monoclonal antibody (MoAb), whereas this concentration of antibody partially inhibited adhesion to alpha 1(I)CB7 and alpha 1(I)CB8. At higher concentrations (3 micrograms/mL) the anti-glycoprotein Ia (GPIa) antibody completely inhibited adhesion to alpha 1(I)CB8 and further reduced adhesion to alpha 1(I)CB7. Platelet adhesion to alpha 1(I)CB3, alpha 1(I)CB7, and alpha 1(I)CB8 was strongly inhibited by an anti-GPIb MoAb. A MoAb against the GPIb-binding site of von Willebrand factor (vWF) strongly inhibited platelet adhesion to alpha 1(I)CB7 and alpha 1(I)CB8, whereas platelet adhesion to alpha 1(I)CB3 was not inhibited. We conclude that under flow conditions alpha 1(I)CB3, alpha 1(I)CB7, and alpha 1(I)CB8 support GPIa/IIa-dependent platelet adhesion. The GPIb-vWF interaction is important under flow conditions for adhesion to alpha 1(I)CB7 and alpha 1(I)CB8 and probably also to alpha 1(I)CB3.

OBJECTIVE--To determine whether the finding of antiphospholipid antibodies in patients with venous thromboembolic episodes should influence the duration of treatment with anticoagulant drugs by mouth. METHODS--A retrospective study was carried out in 19 patients with antiphospholipid antibodies and a history of venous thromboembolic episodes. The median follow up from the first venous thromboembolic episode was 93 months and the median age at this episode was 26 years. The patients had in total 34 venous thromboembolic episodes. The total follow up period comprised 32 periods with and 23 periods without anticoagulant drugs. RESULTS--The probability of being free of recurrent venous thromboembolic episodes, calculated by the Kaplan-Meier method, was significantly influenced by the use of anticoagulant drugs. Patients receiving oral anticoagulants had at eight years a 100% probability of survival without recurrence, whereas patients in whom anticoagulant drugs were stopped had a 50% probability of a recurrent venous thromboembolic episode at two years, and a 78% probability of recurrence at eight years. CONCLUSION--Patients with venous thromboembolic episodes and antiphospholipid antibodies have a high risk for recurrent venous thromboembolic episodes and long term treatment with anticoagulant drugs by mouth is an effective prophylaxis.

The effect of D-Arg-Gly-Asp-Trp (dRGDW), a synthetic RGD-containing peptide, on platelet adhesion and aggregate formation on various purified adhesive proteins and the extracellular matrix of endothelial cells was investigated with anticoagulated blood recirculating through a parallel-plate perfusion chamber. Aggregate formation on the extracellular matrix of phorbol myristate acetate (PMA)-stimulated endothelial cells and on collagen type I was more strongly inhibited by dRGDW at higher shear rates than at a low shear rate. Platelet adhesion to the extracellular matrix of nonactivated and PMA-stimulated endothelial cells was inhibited by dRGDW, especially at high shear rates, probably as a consequence of the inhibition of platelet spreading. Inhibition by dRGDW of platelet adhesion to von Willebrand factor, fibronectin, and fibrinogen was almost complete, indicating that platelet adhesion to these substrates is mediated through RGD-directed receptors. Platelet adhesion to laminin was not inhibited by the peptide, whereas platelet adhesion to collagen was increased as a consequence of the inhibition of aggregate formation. Our results show that dRGDW is a strong inhibitor of platelet adhesion and aggregate formation, especially at high shear rates.

The development of antibodies to factor VIII (inhibitors) in response to clotting-factor concentrates administration in hemophilia is common during the first few years of treatment but rare in multitransfused patients. We have investigated the possible association of a recently introduced factor VIII concentrate (Factor VIII CPS-P) in The Netherlands with the occurrence of inhibitors. To this effect, we conducted two studies. First, we performed a national multicenter study in which clinical information and inhibitor test results were obtained for 447 hemophilia A patients over the period 1988 through 1991. Secondly, for a baseline comparison we estimated the frequency of inhibitor development in a closely followed cohort of 144 patients, from 1984 through 1989. Before the introduction of Factor VIII CPS-P, the incidence of new inhibitors was 4.4/1,000 patient-years in the national study from March 1988 through May 1990, and 3.9/1,000 patient- years in the cohort followed from 1984 through 1989. These figures are similar to the incidence of new inhibitors that was found in a large cohort of patients in the United States followed in the 1970s. In the period that the new concentrate Factor VIII CPS-P was on the market, from June 1990 through November 1991, 11 clinically relevant inhibitors were detected, which yielded an incidence over this interval of 20.1/1,000 patient-years, a 4.5-fold increase compared with the previous interval (C195: 1.4 to 14.3). Nine of these 11 patients had in their lifetime received over 250 infusions with factor VIII preparations. whereas all of the inhibitors detected in the previous time interval, and all of the 24 inhibitor patients described in the US study, had received less than 250 infusions in their lifetime. All patients who developed inhibitors after June 1990 had been exposed to Factor VIII CPS-P, whereas only 75% of the patients who did not develop an inhibitor had been exposed to this product. In a prospective extension of the study, with a second inhibitor measurement after 3 months, we found that one additional inhibitor had developed during 52.5 patient-years of Factor VIII CPS-P use. In conclusion, there has been a sudden increase in the frequency of inhibitor patients, for a large part among multitransfused patients. It seems more than likely that this increase is associated with the introduction of a new factor VIII concentrate in The Netherlands.(ABSTRACT TRUNCATED AT 400 WORDS)

During platelet secretion granule membrane glycoproteins are translocated to the plasma membrane. We report here the biochemical and immunohistochemical characterization of a panel of platelet-secretion-specific, CD62 and CD63 monoclonal antibodies (MoAb), which we raised to thrombin-activated platelets. The CD62 MoAb identify the α-granule membrane protein GMP-140, also designated platelet activation-dependent granule external membrane protein (PADGEM). The number of epitopes on thrombin-activated platelets ranged from 15000 to 20000. The CD63 MoAb recognize a 30–60 kDalton integral membrane protein of lysosomes. Due to its distinct localization, we have designated the CD63 antigen lysosome integral membrane protein, CD63 (LIMP-CD63). The number of epitopes on thrombin-activated platelets ranged from 9000 to 11000. Expression of GMP-140, a member of the Selectin family (also referred as the LEC-CAM family) of adhesion molecules, and LIMP-CD63 was examined on human spleen, thymus and lymph node by immunohistochemistry. Both GMP-140 and LIMP-CD63 showed a wide distribution in lymphoid tissues; vascular endothelial cells and tissue compartments that were readily accessible to blood-borne components were uniformly positive for GMP-140 and LIMP-CD63. Furthermore, LIMP-CD63 was expressed in polymorphonuclear granulocytes and macrophages.

PURPOSE We used alternative chemotherapy immediately followed in early-response patients by high-dose chemoradiotherapy and autologous bone marrow transplantation (ABMT) to treat patients with non-Hodgkin's lymphoma (NHL) who had failed to respond to first-line chemotherapy. PATIENTS AND METHODS Thirty-one patients with NHL of intermediate- or high-grade malignancy who had failed to respond to first-line cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) chemotherapy were treated. Seventeen patients had primary refractory disease and 14 had relapsed from first complete response (CR). The treatment consisted of prednisone, methotrexate, doxorubicin, cyclophosphamide, etoposide, mechlorethamine, vincristine, and procarbazine (ProMACE-MOPP) salvage chemotherapy, followed, in case of responsive disease (at least partial response [PR]), by high-dose cyclophosphamide and total-body irradiation (TBI) with ABMT. RESULTS Twenty-eight of 31 (90%) patients achieved PR (23 patients) or CR (five patients) with ProMACE-MOPP, and three failed to respond. Seventeen of 28 (61%) patients who responded underwent the ABMT procedure, which resulted in CR in 14 patients (82%); three failed to respond. Eleven responsive patients were not transplanted because of residual bone marrow infiltration (five patients), patient refusal (four patients), and ProMACE-MOPP-related mortality (two patients). To date, nine patients are alive and in CR, seven with a median follow-up of 41 months (range, 17 to 84 months). Referring to the original CHOP treatment, five of 17 (29%) patients with primary refractory disease remain free of disease at a median of 36 months after ABMT, and four of 14 (29%) patients in first relapse remain free of disease at a median of 33 months after ABMT. One patient died of AMBT-related toxicity. CONCLUSION ProMACE-MOPP salvage chemotherapy produces a high early-response rate in patients who fail to respond to first-line CHOP, and more than half of the responding patients can be scheduled to receive ABMT, resulting in disease-free survival (DFS) at 3 years in 50% of the transplanted patients and in 25% of the original number of patients intended to receive this treatment.

To identify antigens on the platelet plasma membrane that are exposed after activation, we developed a monoclonal antibody (MoAb) designated RUU-SP 1.77. The RUU-SP 1.77 antigen is present on the membrane of resting platelets at a basal level and is strongly expressed on the plasma membrane after thrombin activation. Freshly fixed platelets bound 4,150 +/- 1,935 (mean +/- SD) RUU-SP 1.77 molecules per platelet; on fixed thrombin-stimulated platelets the number of binding sites was upregulated to 19,050 +/- 5,120 (kd 4.5 +/- 0.8 nmol/L). MoAb RUU-SP 1.77 recognized a major protein of 33 Kd and a minor 28-Kd protein, both under nonreduced and reduced conditions. Immunoelectron microscopic studies showed the presence of the protein associated with the membrane of alpha-granules. Due to the localization associated with the alpha-granule membrane, we have designated it GMP-33 (granule membrane protein with a molecular weight of 33 Kd). Based on structural properties, we conclude that GMP-33 is a protein associated with the alpha-granule membrane that has not been described before.

In a prospective double-blind trial, we treated 194 patients with acute venous thromboembolism with heparin or low molecular weight heparin (LMWH; Fragmin). To evaluate the most important prognostic factors for bleeding, the presenting clinical features of the patients, the patients' anticoagulant responses, and the doses of the drugs were analyzed using univariate and multivariate regression analyses. No significant differences in clinical risk factors associated with bleeding were observed between heparin and LMWH. The univariate analyses ranked the parameters in the following order of importance: World Health Organization (WHO) performance status, history of bleeding tendency, cardiopulmonary resuscitation, recent trauma or surgery, leukocyte counts, platelet counts, duration of symptoms, and body surface area. Patients with WHO grade 4 had an eightfold increase in risk of bleeding as compared with WHO grade 1. Assessment of the individual contribution of each variable using multivariate regression analysis showed that the WHO performance status was the most important independent factor predicting major bleeding. A history of a bleeding tendency, recent trauma or surgery, and body surface area were also independent risk factors. The risk of bleeding was influenced by two factors related to the treatment, the patient's anticoagulant response as measured with the anti-Xa assay and the dose of the drug expressed as U/24 h/m2. An increased risk of bleeding was only observed at mean anti-Xa levels greater than 0.8 U/mL for both drugs. Significantly more major bleedings occurred in patients treated with high doses of the drugs, an observation that was independent of the concomitant anti-Xa levels. It should be considered whether choosing an appropriate initial dose adapted to the patient's body surface area and clinical risk factors can improve the efficacy to safety ratio of heparin treatment.

In a prospective double-blind trial, we treated 194 patients with acute venous thromboembolism with heparin or low molecular weight heparin (LMWH; Fragmin). To evaluate the most important prognostic factors for bleeding, the presenting clinical features of the patients, the patients' anticoagulant responses, and the doses of the drugs were analyzed using univariate and multivariate regression analyses. No significant differences in clinical risk factors associated with bleeding were observed between heparin and LMWH. The univariate analyses ranked the parameters in the following order of importance: World Health Organization (WHO) performance status, history of bleeding tendency, cardiopulmonary resuscitation, recent trauma or surgery, leukocyte counts, platelet counts, duration of symptoms, and body surface area. Patients with WHO grade 4 had an eightfold increase in risk of bleeding as compared with WHO grade 1. Assessment of the individual contribution of each variable using multivariate regression analysis showed that the WHO performance status was the most important independent factor predicting major bleeding. A history of a bleeding tendency, recent trauma or surgery, and body surface area were also independent risk factors. The risk of bleeding was influenced by two factors related to the treatment, the patient's anticoagulant response as measured with the anti-Xa assay and the dose of the drug expressed as U/24 h/m2. An increased risk of bleeding was only observed at mean anti-Xa levels greater than 0.8 U/mL for both drugs. Significantly more major bleedings occurred in patients treated with high doses of the drugs, an observation that was independent of the concomitant anti-Xa levels. It should be considered whether choosing an appropriate initial dose adapted to the patient's body surface area and clinical risk factors can improve the efficacy to safety ratio of heparin treatment.

The pathogenic effect of cytomegalovirus (CMV) infection on the hematopoietic recovery after bone marrow transplantation (BMT) was retrospectively studied in 87 recipients of (nonpurged) autologous BMT and in 56 recipients of allogeneic BMT from HLA-identical siblings. Indications for autologous BMT were lymphomas or acute leukemias and for allogeneic BMT various malignancies or aplastic anemia. Patients were divided for the study in two groups, CMV-positive and CMV-negative on the basis of the CMV status pretransplant, and CMV-negative patients were kept CMV-negative by the local transfusion policy. In allogeneic BMT recipients, platelet recovery was significantly slower in CMV- positive patients than in CMV-negative patients (platelets greater than 50,000 cells/microL after 41 days v 27 days, P = .007). This difference held true when patients with acute graft-versus-host disease above grade I were excluded (platelets greater than 50,000 cells/microL after 42 days v 24 days, P = .01). In autologous BMT, the negative effect on platelet recovery was present in patients with lymphomas, but absent in patients with acute leukemias. Patients with acute leukemias had a very delayed recovery of platelets and granulocytes after autologous BMT, irrespective of the CMV status, probably due to the original stem cell disorder. Platelet recovery was significantly slower in CMV-positive autologous BMT recipients with lymphomas than in those not infected (platelets greater than 50,000 cells/microL after 36 days v 24 days, P = .0002). The presence of CMV infection had no effect on the recovery of granulocytes in autologous or allogeneic BMT. These data show that CMV infection causes delayed platelet recovery after BMT; however, in autologous BMT, the underlying disease (ie, acute leukemia) is more determinant for hematopoiesis after BMT.

Binding of anti-phospholipid antibodies to circulating platelets and its consequences on platelet activation and aggregation was investigated in 11 patients with anti-phospholipid antibodies. Seven patients had mild thrombocytopenia. Nine healthy donors served as controls. Binding to platelets was investigated by performing enzyme- linked immunosorbent assays (ELISAs) with phospholipids as antigen on platelet eluates. Platelet activation was measured by flow cytofluorometry using monoclonal antibodies to an activation-specific lysosomal membrane protein. Findings in ELISA were compared with results of a conventional immunofluorescence method to detect platelet autoantibodies. In seven patients antibodies to negatively charged phospholipids were present in platelet eluates. In all thrombocytopenic patients and controls the platelets were not activated and aggregation was not impaired. There was a positive concordance of 50% between the results of immunofluorescence and ELISA. No apparent relation was found between the results of ELISA or immunofluorescence and platelet counts. It is concluded that anti-phospholipid antibodies can bind to circulating platelets. This binding is not associated with measurable aggregation abnormalities nor with platelet activation characterized by exposure of lysosomal membrane proteins. More studies are necessary to determine the exact role of anti-phospholipid antibodies in the pathogenesis of thrombocytopenia and thrombosis.

Binding of anti-phospholipid antibodies to circulating platelets and its consequences on platelet activation and aggregation was investigated in 11 patients with anti-phospholipid antibodies. Seven patients had mild thrombocytopenia. Nine healthy donors served as controls. Binding to platelets was investigated by performing enzyme- linked immunosorbent assays (ELISAs) with phospholipids as antigen on platelet eluates. Platelet activation was measured by flow cytofluorometry using monoclonal antibodies to an activation-specific lysosomal membrane protein. Findings in ELISA were compared with results of a conventional immunofluorescence method to detect platelet autoantibodies. In seven patients antibodies to negatively charged phospholipids were present in platelet eluates. In all thrombocytopenic patients and controls the platelets were not activated and aggregation was not impaired. There was a positive concordance of 50% between the results of immunofluorescence and ELISA. No apparent relation was found between the results of ELISA or immunofluorescence and platelet counts. It is concluded that anti-phospholipid antibodies can bind to circulating platelets. This binding is not associated with measurable aggregation abnormalities nor with platelet activation characterized by exposure of lysosomal membrane proteins. More studies are necessary to determine the exact role of anti-phospholipid antibodies in the pathogenesis of thrombocytopenia and thrombosis.

To clone the CD63 antigen, originally described as a blood platelet activation marker, we adapted the expression cloning procedure of Seed and Aruffo (Seed, B., and Aruffo, A. (1987) Proc. Natl. Acad. Sci. U.S. A. 84, 3365-3369) to allow cloning of intracellular antigens. A megakaryocyte expression cDNA library was transiently transfected into MOP-8 mouse fibroblasts cultured on polyvinylidene difluoride membranes. Individual cells expressing intracellular CD63 were identified by autoradiography. cDNA was extracted from positive spots and reintroduced into Escherichia coli. After two screening rounds, a CD63 cDNA clone was isolated as assessed by immunofluorescence and Western blot analysis. The single long open reading frame of 238 amino acids contained four putative transmembrane regions and three N-glycosylation sites. The CD63 gene was expressed in a wide variety of cells. Surprisingly, CD63 was identical to ME491, an antigen reported as a melanoma-associated antigen (Hotta, H., Ross, A. H., Huebner, K., Isobe, M., Wendeborn, S., Chao, M. V., Ricciardi, R. P., Tsujimoto, Y., Croce, C. M., and Koprowski, H. (1988) Cancer Res. 48, 2955-2962). By immunoelectron microscopy, co-localization with the lysosomal glycoproteins lamp-1 and -2 identified CD63 as a novel lysosomal membrane glycoprotein. CD63 was not related to the lysosomal glycoprotein family but contained the putative lysosomal targeting signal Gly-Tyr in its short cytoplasmic tail.

This report describes a new variant of hereditary macrothrombocytopenia combined with the presence of neutrophil inclusions that differ from those found in patients with May-Hegglin anomaly, the Chediak-Higashi syndrome or individuals with septicaemia and toxic Döhle bodies in polymorphonuclear leukocytes (PMN). The PMN inclusions in the family described in this report are similar to those found in patients with the Fechtner syndrome, a variant of Alport's syndrome. However, other features of Alport's syndrome, including high frequency deafness, congenital cataracts, and chronic interstitial nephritis are absent in the members of the family described here. We have named this anomaly the Sebastian platelet syndrome. The macrothrombocytopenia and neutrophil inclusions observed in this family can occur in the absence of other congenital anomalies and therefore represent a unique syndrome.

Acetylsalicylic acid (aspirin) induces an irreversible inactivation of cyclo-oxygenase in blood platelets which lasts for the entire period that the platelets remain in the circulatory system, 7 to 10 days. In order to prevent excessive bleeding, patients presenting for surgery are asked to stop using aspirin 10 days before the procedure. Some studies have found that aspirin causes increased peri-operative blood loss, whilst other studies have found that it does not. All effect studies found in Medline (January 1966-May 2002) on surgery and bleeding complications due to aspirin were analysed. The studies available were assessed for methodological quality and the results were summarised in an evidence table. No clinically relevant bleeding complications were reported for cardiovascular, vascular and orthopaedic surgery and epidural anaesthesia. Most studies reported an increase in clinically non-relevant bleeding induced by aspirin. The literature contains too little information on cataract surgery, dermatological surgery, gynaecological and abdominal surgery, ENT and dental surgery, urological surgery, lung biopsy and endoscopic biopsy. In those types of surgery in which even a minor bleeding leads to severe complications, e.g. neurosurgery, aspirin should be withdrawn 5-10 days in advance. Also in patients with coagulation disorders, aspirin should be withdrawn prior to the operation. There is no scientific evidence for the withdrawal of aspirin in all patients, 5-10 days prior to surgery. Indeed for heart patients in particular, the continued use of aspirin is recommended.

Background— Serial ultrasonography is reliable for the diagnosis of deep venous thrombosis in symptomatic patients, but the low prevalence of thrombosis in this group renders the approach costly and inconvenient to patients. We studied the clinical validity of the combination of a pretest clinical probability score and a D-dimer test in the initial evaluation of patients suspected of deep venous thrombosis. Methods and Results— Patients with a normal D-dimer concentration ( Conclusion— The combination of a non-high pretest clinical probability score and a normal D-dimer concentration is a safe strategy to rule out deep venous thrombosis and to withhold anticoagulation.

Pyruvate kinase (PK) deficiency is a common cause of hereditary non-spherocytic haemolytic anaemia. It is an autosomal recessive disorder caused by mutations in the gene coding for erythrocyte and liver-type pyruvate kinase (PKLR). So far, more than 130 mutations in this gene have been identified. Clinical symptoms, usually restricted to homozygous and compound-heterozygous individuals, are variable, ranging from neonatal jaundice requiring erythrocyte transfusions to a fully compensated haemolytic anaemia. The exact mechanism of erythrocyte destruction is unknown, however adenosine-triphosphate depletion and an increase in 2,3-disphosphoglycerate are thought to be important. The diagnosis of pyruvate kinase deficiency depends upon the demonstration of low PK enzyme activity. Due to the pitfalls in determining true PK activity, DNA testing is a valuable tool in the diagnosis of pyruvate kinase deficiency. By centralizing the molecular diagnostics of pyruvate kinase deficiency in Utrecht, more care can be provided for the diagnosis, treatment and support of patients.

TAFI (thrombin activatable fibrinolysis inhibitor) down regulates fibrinolysis after activation by relatively high concentrations of thrombin generated during coagulation via thrombin mediated factor XI activation and subsequent activation of the intrinsic pathway. It is this secondary burst of thrombin that is severely diminished in haemophilia A, a deficiency of coagulation factor VIII. We therefore investigated the role of TAFI in haemophilia A by measuring the clot lysis times of tissue factor induced fibrin formation and tPA mediated fibrinolysis. In haemophilia A plasma clot lysis times were normal at relatively high tissue factor concentrations but severely decreased at moderate to low tissue factor concentrations, indicating that the thrombin generation via the extrinsic pathway was insufficient to activate TAFI. Addition of factor VIII, TAFI or thrombomodulin restored the clot lysis times at low tissue factor concentrations. This confirms the hypothesis that the bleeding disorder in haemophilia A is not merely a defect in the initial clot formation but is in fact a triple defect: reduced thrombin formation via the extrinsic pathway at low tissue factor concentrations, a reduced secondary burst of thrombin generation via the intrinsic pathway and a defective down regulation of the fibrinolytic system by the intrinsic pathway.

GMP-33 is a platelet membrane associated protein that is recognised by RUU-SP 1.77, an antibody raised against activated platelets. GMP-33 is predominantly associated with the membrane of platelet alpha-granules and it is translocated to the plasma membrane upon platelet activation (Metzelaar et al. Blood 1992; 79: 372-9). In this study we have isolated the protein by immunoaffinity chromatography. The N-terminus was sequenced and was identical to the N-terminal sequence of human thrombospondin. The protein was N-glycosylated and bound to heparin as would be expected of the N-terminal part of thrombospondin. RUU-SP 1.77 reacted only with reduced thrombospondin. Plasmin and trypsin digestion of thrombospondin yielded fragments of approximately the same size as GMP 33 that reacted with RUU-SP 1.77 after reduction. No evidence for alternative splicing was found. We postulate that GMP 33 is an N-terminal proteolytic fragment of thrombospondin that is membrane associated.

The success of allogeneic bone marrow transplantation (BMT) is limited by the major complications, graft-versus-host disease (GVHD) and relapse. The very beneficial effect of maximal T-cell depletion of the graft for prevention of GVHD has been counterbalanced by an increase in graft failure and relapse of disease. Therefore, we started an approach of partial T-cell depletion of the graft. GVHD and graft-versus-leukaemia (GVL) are strongly correlated after non-T cell-depleted BMT. Here, we report whether the correlation between GVHD and GVL also exists in partial T cell-depleted BMT from sibling donors. We retrospectively studied 117 adult patients with early haematological malignancies. Our method of partial T-cell depletion gave a relapse rate in patients with acute leukaemias similar to that observed in non-T cell-depleted BMT. However, patients with chronic myeloid leukaemia had a relapse rate that was similar to that observed in maximal T cell-depleted BMT. We found a significant correlation between the presence of chronic GVHD and an improved disease-free survival. Nevertheless, overall survival was lower in patients with chronic GVHD. There was no correlation between the occurrence of acute GVHD and disease-free or overall survival.

The development of arthropathy is a serious complication of severe haemophilia. With the use of prophylaxis, bleeds can be prevented and arthropathy delayed. We investigated whether an individually tailored prophylactic regimen can prevent arthropathy and whether it had a similar effect on orthopaedic outcome compared with that of a high-dose regimen. Efficacy was determined clinically and by radiographs of six major joints. Prophylaxis was started in 70 patients at a mean age of 4.1 years. Mean follow-up was 15.6 years (range 8-24.5 years). The mean factor VIII consumption was 2319 IU/kg/year. The mean number of joint bleeds was 3.5/year and the mean clinical score (maximum score 90) was 1.0, with a mean Pettersson joint score (maximum score 78) of 3.0 at a mean age of 13.5 years. In conclusion, long-term, early-onset, individualized prophylaxis in haemophilia is feasible and prevents arthropathy.

Acute promyelocytic leukaemia (APL) is a disease that is distinguished from other leukaemias by the high potential for early haemorrhagic death. Several processes are involved, such as disseminated intravascular coagulation and hyperfibrinolysis. Recently, TAFI (thrombin-activatable fibrinolysis inhibitor) was identified as a link between coagulation and fibrinolysis. TAFI can be activated by thrombin, and in its activated form potently attenuates fibrinolysis by removing C-terminal lysine and arginine residues that are important for the binding and activation of plasminogen. Activation of TAFI by the coagulation system results in a down-regulation of fibrinolytic activity and, thereby, prevents a rapid dissolution of the fibrin clot. To establish whether TAFI was involved in the severity of the bleeding complications in APL, the TAFI antigen and activity levels were determined in a group of 15 patients. The TAFI antigen concentration was normal, but the activity of TAFI was severely reduced in APL by approximately 60%. The reduction of TAFI activity was most probably caused by the action of plasmin on TAFI because in vitro experiments revealed that plasmin slightly reduced antigen levels but severely reduced TAFI activity. The acquired functional TAFI deficiency in APL may contribute to the severity of the haemorrhagic diathesis because of the impaired capacity of the coagulation system to protect the fibrin clot from fibrinolysis.

Abstract —LDL is known to increase the sensitivity of human platelets for agonists and to induce aggregation and secretion independently at high concentrations, but its mechanism of action is largely obscure. To clarify how LDL increases platelet sensitivity, cells were incubated in lipoprotein-poor plasma and treated with collagen at a concentration that induced ≈20% secretion of 14 C-serotonin. Preincubation with LDL (30 minutes at 37°C) enhanced secretion in a dose-dependent manner to 60±14% at a concentration of 2 g LDL protein/L. Similar stimulation by LDL was seen when secretion was induced by the thrombin receptor–activating peptide. This enhancement was strongly reduced (1) in the presence of monoclonal antibody PAC1 against activated α IIb β 3 , a polyclonal antibody against α IIb , and in the presence of the fibrinogen peptides GRGDS and HHLGGAKQAGDV; (2) in α IIb β 3 -deficient platelets; and (3) after dissociation of α IIb β 3 . In contrast, binding of 125 I-LDL to normal platelets in the presence of PAC1, anti-α IIb , GRGDS, and HHLGGAKQAGDV, and to α IIb β 3 -deficient platelets was normal. LDL increased the surface expression of fibrinogen in lipoprotein-poor plasma and fibrinogen-free medium, suggesting that extracellular and granular fibrinogen bind to α IIb β 3 after platelet-LDL interaction. Platelets deficient in fibrinogen (IIb β 3 preserved responsiveness to LDL, indicating that occupancy of α IIb β 3 was not restricted to fibrinogen. Inhibition of protein kinase C (bisindolylmaleimide) diminished fibrinogen binding and sensitization by LDL; inhibition of tyrosine kinases (herbimycin A) left fibrinogen binding unchanged but diminished sensitization by LDL. We conclude that an increased concentration of LDL, such as observed in homozygous familial hypercholesterolemia, sensitizes platelets to stimulation by collagen and thrombin receptor–activating peptide via ligand-induced outside-in signaling through integrin-α IIb β 3 .

Im Jahr 1967 beschrieben unabhangig voneinander Hardisty u. Hutton sowie Weiss eine Storung in der Sezernierung von Thrombozyteninhalts-stoffen bei Patienten mit einer angeborenen Blutungsneigung. Die beschriebenen Patienten zeigten eine Storung der thrombozytaren ADP (Adenosindiphosphat)-Freisetzung sowie eine I verminderte Kollagen-induzierte Thrombozytenaggregation. Aufgrund dieser Befunde wurde eine Storung in der Freisetzung und/oder der Bildung des intrathrombozytar gespeicherten ADP-Pools („storage pool“) postuliert. Als anatomisches Korrelat dieser vermuteten Speicherstorung wurde im Jahr 1970 von Holmsen u. Weiss in Thrombozyten von Patienten mit einer Storage-Pool-Erkrankung eine Verminderung der ADP-speichernden „dense bodies“ (dichte Granula) beschrieben. Die Storung in der ADP-Speicherung fuhrte auch zur ursprunglichen Krankheitsbezeichnung „storage pool disease“. In der Folge wurden ahnliche Befunde auch bei Patienten mit anderen angeborenen thrombozytaren Erkrankungen, wie dem Hermansky-Pudlak-Syndrom und dem Chediak-Higashi-Syndrom, sowie bei erworbenen Thrombozytopathien, wie sie im Rahmen von myeloproliferativen Erkrankungen oder bei Patienten mit disseminierter intravasaler Gerinnung auftreten, beobachtet. Diese bei thrombozytaren Erkrankungen unterschiedlichster Genese auftretende einheitliche pathophysio-logische Symptomatik zeigt, das die Storage-Pool-Erkrankung kein homogenes Krankheitsbild darstellt, sondern als einheitliches Symptom unterschiedlicher thrombozytarer Erkrankungen aufgefast werden mus. Dementsprechend wurde die Bezeichnung der „storage pool deficiency (SPD)“ gepragt. Der ursprunglich auf die Beschreibung einer Storung der Sezernierungsreaktion der „dense bodies“ begrenzte Begriff der SPD wurde, nachdem eine ahnliche Symptomatik auch fur andere thrombozytare Speicherorganellen beschrieben worden war, auf alle Storungen ausgedehnt, die durch eine Verringerung der in den thrombozytaren Speicherorganellen gespeicherten Substanzen gekennzeichnet sind. Inzwischen werden 3 verschiedene Formen des SPD unterschieden: die (d-SPD bezeichnet eine isolierte Verminderung der „dense bodies“ und ihrer Inhaltsstoffe, die α-SPD bezeichnet eine isolierte Verminderung der α-Granula und ihrer Inhaltsstoffe, wahrend die αd-SPD eine Kombination beider Defekte darstellt. Ein Mangel an thrombozytaren Lysosomen ist bisher beim Menschen noch nicht beschrieben worden.

Background and Purpose Activation of endothelial cells and platelets plays an important role in the development of atherosclerosis and thrombotic disorders. Soluble adhesion molecules originating from these cells can be demonstrated in plasma. We hypothesized that elevated plasma concentrations of soluble P-selectin (sP-selectin), soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1), and soluble E-selectin (sE-selectin) can reflect activation of endothelial cells and/or platelets in acute ischemic stroke and in previously symptomatic internal carotid artery stenosis. Methods Plasma was sampled from patients within 2 days of acute ischemic stroke (n=28), from patients with a previous (>1 week) transient or persistent ischemic neurological deficit associated with stenosis of the internal carotid artery (n=34), and from control patients without a history of vascular disease (n=34). Concentrations of sP-selectin, sICAM-1, sVCAM-1, and sE-selectin were measured by means of an enzyme-linked immunosorbent assay. Results Compared with control subjects, sP-selectin and sE-selectin were significantly elevated in the acute stage of ischemic stroke ( P P =.001, respectively) as well as in previously symptomatic carotid stenosis ( P P =.0007). sICAM-1 and sVCAM-1 were not increased. Conclusions The elevated levels of sE-selectin indicate that endothelial cell activation occurs both in the acute stage of ischemic stroke and in previously symptomatic carotid atherosclerosis. Increased sP-selectin concentrations reflect endothelial cell activation as well but may also be caused by platelet activation.

Patients with recurrent or refractory low-grade non-Hodgkin's lymphoma (NHL) are increasingly treated with myeloablative therapy and autologous stem cell transplantation. However, allogeneic bone marrow transplantation (BMT) is only sporadically performed in such patients. Therefore, we wish to compare treatment results of patients with recurrent or refractory low-grade NHL who underwent allogeneic BMT with those who underwent autologous BMT in our center. Twenty-eight patients were studied. The patients had received 2 to 5 lines of conventional chemotherapy before the BMT procedure. Eighteen patients, all with chemotherapy-sensitive disease at the time of transplantation, underwent autologous BMT and 10 patients, of whom 7 with chemotherapy-resistant disease at the time of transplantation, underwent allogeneic BMT. Furthermore, all allogeneic BMT patients had overt lymphoma infiltration of the BM at the time of transplantation. The conditioning regimen consisted of cyclophosphamide plus total body irradiation in all 28 patients. All allogeneic BMT patients achieved complete remission, 3 patients had a treatment-related death, and 7 patients are alive and disease-free with a median follow-up of 41 months. In contrast, none of the autologous BMT patients died of transplant-related complications. However, despite the fact that all autologous BMT patients had chemotherapy-sensitive disease and partial remission was converted to complete remission by the BMT procedure in 67% of them, only 3 of 18 patients are alive and disease-free. The probability of relapse or disease-progression among allogeneic BMT patients was 0% compared with 83% for autologous BMT patients (P = .002). Progression-free survival rates 2 years after BMT were 68% for allogeneic BMT patients and 22% for autologous BMT patients (P = .049). Although the numbers of patients are small, this study suggests that allogeneic BMT offers a better chance for cure than autologous BMT for patients with poor-prognosis low-grade lymphoma, and the difference in relapse or disease progression is strongly suggestive for the existence of a graft-versus-low-grade lymphoma effect.

P-selectin is a 140 kD protein found in the alpha-granules of platelets and the Weibel-Palade bodies of endothelial cells. On cell activation it is expressed on the cell surface and also secreted into plasma. Whether the circulating soluble P-selectin (sP-selectin) originates from platelets, endothelial cells, or both, is not known. We studied the level of sP-selectin in diseases with different platelet counts, with or without evidence of endothelial cell activation. Endothelial cell activation was confirmed by the detection of sE-selectin and ED1-fibronectin. A significant positive correlation between platelet count and sP-selectin concentration was observed in healthy controls, and in patients with thrombocytopenia due to bone marrow aplasia, or with thrombocytosis (r = 0.85; n = 47; p0.001). In patients with idiopathic thrombocytopenic purpura (ITP) the sP-selectin concentration was 110 +/- 39 ng/ml (n = 10), compared to 122 +/- 38 ng/ml in healthy controls (n = 26). However, their mean platelet count was lower (58 x 10(9)/l versus 241 x 10(9)/l in the control group). Accordingly, the levels of sP-selectin expressed per platelet increased to significantly higher levels (2.0 +/- 1.2 versus 0.6 +/- 0.2 fg/platelet in the control group; p0.0001). This suggests increased platelet turnover in patients with ITP. High levels of sP-selectin were found in patients with sepsis (398 +/- 203 ng/ml; n = 15) and with thrombotic thrombocytopenic purpura (TTP; 436 +/- 162 ng/ml; n = 12). Compared with patients with ITP, the concentration of sP-selectin per platelet was higher in patients with sepsis (4.8 +/- 4.3 fg/platelet; p0.005) or TTP (17.1 +/- 9.5 fg/platelet; p0.001). Endothelial cells are very likely to be the source in these patients and the presence of endothelial cell activation was confirmed by increased levels of circulating E-selectin and ED1-fibronectin. This study suggests that platelets are the major source of circulating sP-selectin in healthy individuals. Endothelial cell activation is associated with an increased sP-selectin concentration per platelet.