Hermansky-Pudlak syndrome is caused by mutations in HPS4, the human homolog of the mouse light-ear gene.

Hermansky-Pudlak syndrome (HPS) is a disorder of organelle biogenesis in which oculocutaneous albinism, bleeding and pulmonary fibrosis result from defects of melanosomes, platelet dense granules and lysosomes. HPS is common in Puerto Rico, where it is caused by mutations in the genes HPS1 and, less often, HPS3 (ref. 8). In contrast, only half of non-Puerto Rican individuals with HPS have mutations in HPS1 (ref. 9), and very few in HPS3 (ref. 10). In the mouse, more than 15 loci manifest mutant phenotypes similar to human HPS, including pale ear (ep), the mouse homolog of HPS1 (refs 13,14). Mouse ep has a phenotype identical to another mutant, light ear (le), which suggests that the human homolog of le is a possible human HPS locus. We have identified and found mutations of the human le homolog, HPS4, in a number of non-Puerto Rican individuals with HPS, establishing HPS4 as an important HPS locus in humans. In addition to their identical phenotypes, le and ep mutant mice have identical abnormalities of melanosomes, and in transfected melanoma cells the HPS4 and HPS1 proteins partially co-localize in vesicles of the cell body. In addition, the HPS1 protein is absent in tissues of le mutant mice. These results suggest that the HPS4 and HPS1 proteins may function in the same pathway of organelle biogenesis.

Usefulness of a semiquantitative D-dimer test for the exclusion of deep venous thrombosis in outpatients.

PURPOSE: The D-dimer test is used commonly in diagnostic strategies to reduce the need for ultrasonography in patients suspected of having deep venous thrombosis. We studied several clinical and laboratory variables that might limit the accuracy of a semiquantitative D-dimer test. SUBJECTS AND METHODS: In this retrospective cohort study, 704 outpatients suspected of having deep venous thrombosis underwent a semiquantitative D-dimer test and ultrasonography. The performance of the D-dimer test was calculated in patients using anticoagulants (n =61), patients with previous thrombosis (n =127), and patients with malignancy (n =47), including 39 patients with more than one of these characteristics. The 508 remaining patients were considered to be the reference group. RESULTS: A total of 254 patients (36%) had evidence of deep venous thrombosis. The D-dimer test had a sensitivity of 99% (174/176; 95% confidence interval [CI]: 96% to 100%) and a negative predictive value of 98% (98/100; 95% CI: 93% to 100%) in the reference group. The sensitivity of the D-dimer test in patients using oral anticoagulants was 75% (6/8; 95% CI: 35% to 97%; P =0.01 compared with the reference group). Test sensitivity was 96% (51/53; 95% CI: 87% to 100%) in patients with previous thrombosis, and 100% (29/29; 95% CI: 88% to 100%) in patients with cancer. However, 553 (79%) of all patients, including 43 of the cancer patients (91%), had an abnormal D-dimer test. CONCLUSION: The semiquantitative D-dimer test in this study has a high sensitivity and negative predictive value in the exclusion of deep venous thrombosis, except perhaps among patients using oral anticoagulants. D-dimer tests in patients with cancer and in patients over 70 years old may not be worthwhile, because the tests are usually positive.

Enhancement of fibrinolytic potential in vitro by anticoagulant drugs targeting activated factor X, but not by those inhibiting thrombin or tissue factor.

Tissue factor-induced coagulation leads to the generation of a small amount of thrombin, resulting in the formation of a fibrin clot. After clot formation, thrombin generation continues resulting in the activation of thrombin activatable fibrinolysis inhibitor, leading to downregulation of fibrinolysis. In this study, the effect of anticoagulant drugs targeting different steps in the coagulation cascade on clot formation and subsequent breakdown was investigated using a plasma-based clot lysis assay. All drugs tested significantly delayed clot formation; only those drugs targeting activated factor X (FXa) (tissue factor pathway inhibitor, fondaparinux, and low molecular weight heparin) accelerated fibrinolysis. Anticoagulant drugs targeting tissue factor (active site-inactivated recombinant activated factor VII) or thrombin (hirudin and d-phenylalanyl-l-prolyl-l-arginyl chloromethyl ketone) did not affect clot lysis time. In accordance with these findings, it was shown that total thrombin generation, as quantified by the endogenous thrombin potential, was only affected by anticoagulant drugs targeting FXa when all drugs were used in a concentration resulting in doubling of clotting time. Induction of hyperfibrinolysis by anticoagulant drugs directed against FXa might be beneficial as increased clot breakdown might facilitate thrombolysis or prevent re-occlusion. On the other hand, the induction of hyperfibrinolysis by these compounds might increase the risk of bleeding complications.

Molecular basis of congenital afibrinogenaemia in a Dutch family.

Congenital afibrinogenaemia is a rare autosomal recessive disorder characterized by complete absence or trace amounts of fibrinogen. Here we report the identification of the molecular defect underlying afibrinogenaemia in a Dutch patient. DNA sequence analysis of the fibrinogen Aalpha, Bbeta and gamma-genes revealed a homozygous deletion of two adenines between nucleotides 3120 and 3122 in exon 4 of the gene coding for the Aalpha-chain. This deletion results in a frameshift with a predicted premature end of translation at codon 140. This is the first report of a patient homozygous for this rare mutation associated with afibrinogenaemia.

Myeloid sarcoma presenting as a recurrent, multifocal nerve root entrapment syndrome.

BACKGROUND: Myeloid sarcoma is an extramedullary manifestation of haematologic malignancy, most commonly acute myeloid leukemia (AML), which can cause neurological symptoms. CASE DESCRIPTION: A 45-year-old male with a history of AML presented with a lumbosacral nerve root entrapment syndrome followed by cauda equina compression, but without systemic signs of AML recurrence. MRI showed a mass compressing the spinal cord at level L5-S2. After surgically removing the tumour pathologic examination yielded a myeloid sarcoma. Combined chemotherapy and radiation therapy followed. Five months later the patient developed a thoracal (Th10-Th11) radiculopathy due to a relapse of the myeloid sarcoma, followed by C8-Th1-radiculopathy caused by leptomeningeal spread. CONCLUSION: This case forms the first description of recurrent, multifocal and progressive radiculopathy due to myeloid sarcoma. This diagnosis should be considered in patients with radiculopathy with previous haematological malignancy and/or signs or symptoms of such disease; the absence of systemic disease activity does not rule out myeloid sarcoma.

The FcgammaRIIa-polymorphic site as a potential target for acute graft-versus-host disease in allogeneic stem cell transplantation.

Graft-versus-host disease (GVHD) is a serious complication of allogeneic stem cell transplantation for hematologic diseases. Antigen mismatches due to polymorphic differences between the patient and donor are central in the mechanisms of GVHD. Polymorphisms are known for FcgammaRIIa, and this molecule is present on endothelial, dendritic, and Langerhans cells. Donor cells reacting against the patient as GVHD can also react against the malignancy of the patient, and this is known as the graft-versus-leukemia (GVL) effect. Because the FcgammaRIIa molecule is also present on acute myeloid leukemia (AML) cells, an FcgammaRIIa mismatch could be a target for both GVHD and GVL. We retrospectively studied 73 AML patients and analyzed the differences in GVHD and relapse incidence between patients with and without a pro-GVHD/GVL FcgammaRIIa allotype mismatch. We observed a difference in FcgammaRIIa receptors with a pro-GVHD/GVL mismatch in 18 patient/donor pairs (25%). Univariate and multivariate analyses demonstrated the pro-GVHD mismatch to be a significant risk factor for the development of acute GVHD. There was no effect on the occurrence of chronic GVHD. The relapse incidence was not significantly different for patients with or without the pro-GVL mismatch, although there was a trend for fewer relapses in standard-risk AML patients with the pro-GVL mismatch. We conclude that the polymorphism of the FcgammaRIIa receptor may be a candidate target for acute GVHD.

Recombinant factor VIIa enhances deposition of platelets with congenital or acquired alpha IIb beta 3 deficiency to endothelial cell matrix and collagen under conditions of flow via tissue factor-independent thrombin generation.

A novel approach to treat bleeding episodes in patients with Glanzmann thrombasthenia (GT) and perhaps also in patients receiving alpha IIb beta 3 inhibitors is the administration of recombinant factor VIIa (rFVIIa). The mechanism of action of rFVIIa in these patients is, however, still unclear. We studied the effect of rFVIIa-mediated thrombin formation on adhesion of alpha IIb beta 3-deficient platelets under flow conditions. Adhesion of alpha IIb beta 3-deficient platelets to the extracellular matrix (ECM) of stimulated human umbilical vein endothelial cells or to collagen type III was studied using a model system with washed platelets and red cells. When alpha IIb beta 3-deficient platelets were perfused over the surface at arterial shear rate for 5 minutes, a low surface coverage was observed (GT platelets, mean +/- SEM, 37.5% +/- 5.0%; normal platelets preincubated with an RGD-containing peptide, 7.4% +/- 2.1%). When rFVIIa, together with factors X and II, was added to the perfusate, platelet deposition significantly increased (GT platelets, mean +/- SEM, 67.0% +/- 4.3%; normal platelets preincubated with an RGD-containing peptide, 48.2% +/- 2.9%). The same effect was observed when normal platelets were pretreated with the commercially available anti-alpha IIb beta 3 drugs abciximab, eptifibatide, or tirofiban. It was shown that tissue factor-independent thrombin generation (presumably induced by binding of rFVIIa to adhered platelets) was responsible for the increase in platelet deposition. In conclusion, defective adhesion of alpha IIb beta 3-deficient platelets to ECM can be restored by tissue factor-independent rFVIIa-mediated thrombin formation. The enhanced generation of platelet procoagulant surface facilitates fibrin formation, so that lack of platelet aggregate formation might be compensated for.

Factor V Leiden in central venous catheter-associated thrombosis.

Subclavian vein thrombosis is a well-recognized complication following central venous catheter insertion and is associated with significant morbidity. The factor V Leiden mutation is an important risk factor for deep venous thrombosis and pulmonary embolism. Whether this mutation also predisposes patients fitted with a central venous catheter to subclavian vein thrombosis is not known. The occurrence of central venous catheter-associated thrombosis was investigated in 277 consecutive patients receiving an allogeneic bone marrow transplantation. All patients received a tunnelled double or triple catheter positioned in the subclavian vein. Catheter-associated thrombosis was diagnosed on the basis of clinical signs of thrombosis, i.e. swelling and/or redness of the limb or venous engorgement and was confirmed with a colour-flow Doppler ultrasound. Thirteen patients were heterozygous for the factor V Leiden mutation. Seven of these patients had a subclavian vein thrombosis (54%), while this occurred in only 9% of the factor V Leiden-negative patients, corresponding with a relative risk of 7.7 (95% CI 3.3-17.9). Factor V Leiden is attributable for 17.3% of all thrombosis in patients with central venous catheters. The majority of patients with the factor V Leiden mutation with a central venous catheter will develop thrombosis. Patients with a factor V Leiden mutation should receive adequate thrombosis prophylaxis upon catheter introduction and the catheter should be removed immediately after the treatment. Based on this very high risk, we advise testing for factor V Leiden in all bone marrow transplantation patients receiving a central venous catheter.

Low molecular weight heparin (dalteparin) is equally effective as unfractionated heparin in reducing coagulation activity and perfusion abnormalities during the early treatment of pulmonary embolism.

Little is known about the differences between unfractionated heparin (UFH) and low molecular weight heparin (LMWH) with regard to their effects on coagulation activity during treatment for pulmonary embolism. The objective of this study was to compare UFH and LMWH (dalteparin) in the early treatment of pulmonary embolism in terms of control of coagulation markers and perfusion abnormalities. Thirty-seven patients with acute pulmonary embolism were randomized to receive intravenous UFH or subcutaneous dalteparin, each accompanied by acenocoumarol. Daily blood samples were obtained for the measurement of thrombin generation (fragments 1 and 2 [F1+2], thrombin-antithrombin (TAT) complexes and fibrin monomers [FMs]) and fibrinolysis (d-dimer concentrations and clot-lysis times). Ventilation-perfusion scintigraphies were performed, and with the data they yielded, percentage of vascular obstruction scores (PVOs) were calculated on days 0 and 5. The international normalized ratio was within the therapeutic range in both groups on day 3. F1+2 and TAT complexes rapidly normalized, without differences between the groups (P =.5 and.4, respectively). FM levels did not decrease and, in fact, showed an increase in the UFH group from day 3 on (P <.05 between groups). d-Dimer levels decreased over time, with no differences between groups (P =.6). Clot-lysis times were shorter in the UFH group (P <.05). PVOs on days 0 and 5 were not different (P =.5 and.8, respectively), but the decrease in PVOs over time was greater in the dalteparin group (P =.04). These results show that dalteparin is at least as effective as UFH in reducing coagulation activity and perfusion abnormalities in the early treatment of pulmonary embolism.

Inhibition of fibrinolysis by recombinant factor VIIa in plasma from patients with severe hemophilia A.

Recombinant factor VIIa (rFVIIa) is a novel prohemostatic drug for patients with hemophilia who have developed inhibitory antibodies. The postulation has been made that hemophilia is not only a disorder of coagulation, but that hyperfibrinolysis due to a defective activation of thrombin activatable fibrinolysis inhibitor (TAFI) might also play a role. In this in vitro study, the potential of rFVIIa to down-regulate fibrinolysis via activation of TAFI was investigated. rFVIIa was able to prolong clot lysis time in plasmas from 17 patients with severe hemophilia A. The prolongation of clot lysis time by rFVIIa was completely abolished by addition of an inhibitor of activated TAFI. The concentration of rFVIIa required for half maximal prolongation of clot lysis time (C(lys 1/2)-VIIa) varied widely between patients (median, 73.0 U/mL; range, 10.8-250 U/mL). The concentration of rFVIIa required for half maximal reduction of clotting time (C(clot 1/2)-VIIa) was approximately 10-fold lower than the C(lys 1/2)-VIIa value (median, 8.4 U/mL; range, 1.7-22.5 U/mL). Inhibition of TFPI with a polyclonal antibody significantly decreased C(lys 1/2)-VIIa values (median, 2.6 U/mL; range, 0-86.9 U/mL), whereas C(clot 1/2)-VIIa values did not change (median, 7.2 U/mL; range, 2.2-22.5 U/mL). On addition of 100 ng/mL recombinant full-length TFPI, a nonsignificant increase of C(lys 1/2)-VIIa values was observed (median, 119.2 U/mL; range, 12.3-375.0 U/mL), whereas C(clot 1/2)-VIIa values did not change (median, 8.8 U/mL; range, 2.6-34.6 U/mL). In conclusion, this study shows that rFVIIa both accelerates clot formation and inhibits fibrinolysis by activation of TAFI in factor VIII-deficient plasma. However, a large variability in antifibrinolytic potential of rFVIIa exists between patients.

Recombinant factor VIIa improves clot formation but not fibrolytic potential in patients with cirrhosis and during liver transplantation.

Cirrhosis is associated with a bleeding tendency, which is particularly pronounced during orthotopic liver transplantation (OLT). A novel approach to treating the bleeding diathesis of patients with cirrhosis is administration of recombinant factor VIIa (rFVIIa). This study examined whether the efficacy of rFVIIa in cirrhosis might be explained in part by enhanced down-regulation of fibrinolysis by thrombin-activatable fibrinolysis inhibitor (TAFI). Addition of therapeutic or supratherapeutic doses of rFVIIa to plasma of 12 patients with stable cirrhosis did not result in a prolongation of clot lysis time, though clotting times were significantly reduced. Also, clot lysis assays of plasma samples taken during and after OLT, which was performed with or without a single bolus dose of rFVIIa, did not show any effect of rFVIIa on plasma fibrinolytic potential. In conclusion, this study shows no evidence for an antifibrinolytic effect of rFVIIa in cirrhotic patients or in patients undergoing OLT.