H. Karel Nieuwenhuis

A prospective, controlled multicenter study on the obstetric risks of pregnant women with antiphospholipid antibodies

OBJECTIVES A prospective, controlled multicenter study was performed to estimate the obstetric risks of antiphospholipid antibodies (the lupus anticoagulant and anticardiolipin antibodies). In addition, the risks of prior thrombosis, obstetric history, systemic lupus erythematosus, and high-dose prednisone treatment were evaluated. STUDY DESIGN After screening for antiphospholipid antibodies in patients with lupus erythematosus or women with prior fetal loss(es), 59 subsequent pregnancies with and 54 without these antibodies were followed. RESULTS The presence of the lupus anticoagulant and a history of at least three spontaneous abortions could predict fetal loss (p = 0.032 and 0.001, respectively). In live born infants, a low birth weight could be predicted by the presence of anticardiolipin antibodies (p = 0.034), prior intrauterine fetal death (p = 0.025), and treatment with high-dose prednisone (p = 0.002). No relationships were seen between antiphospholipid antibodies and small-for-gestational-age newborns and pregnancy-induced hypertension or preeclampsia. The disappearance of antiphospholipid antibodies during pregnancy was not correlated with live birth. CONCLUSION It is concluded that the presence of antiphospholipid antibodies is a risk factor for adverse pregnancy outcome.

Sequential Regulation of the Small GTPase Rap1 in Human Platelets

ABSTRACT Rap1, a small GTPase of the Ras family, is ubiquitously expressed and particularly abundant in platelets. Previously we have shown that Rap1 is rapidly activated after stimulation of human platelets with α-thrombin. For this activation, a phospholipase C-mediated increase in intracellular calcium is necessary and sufficient. Here we show that thrombin induces a second phase of Rap1 activation, which is mediated by protein kinase C (PKC). Indeed, the PKC activator phorbol 12-myristate 13-acetate induced Rap1 activation, whereas the PKC-inhibitor bisindolylmaleimide inhibited the second, but not the first, phase of Rap1 activation. Activation of the integrin αIIbβ3, a downstream target of PKC, with monoclonal antibody LIBS-6 also induced Rap1 activation. However, studies with αIIbβ3-deficient platelets from patients with Glanzmanns thrombasthenia type 1 show that αIIbβ3 is not essential for Rap1 activation. Interestingly, induction of platelet aggregation by thrombin resulted in the inhibition of Rap1 activation. This downregulation correlated with the translocation of Rap1 to the Triton X-100-insoluble, cytoskeletal fraction. We conclude that in platelets, α-thrombin induces Rap1 activation first by a calcium-mediated pathway independently of PKC and then by a second activation phase mediated by PKC and, in part, integrin αIIbβ3. Inactivation of Rap1 is mediated by an aggregation-dependent process that correlates with the translocation of Rap1 to the cytoskeletal fraction.

Comparing Subcutaneous Danaparoid with Intravenous Unfractionated Heparin for the Treatment of Venous Thromboembolism: A Randomized Controlled Trial

Danaparoid (Org 10172; Organon Scientific Development Group, Oss, the Netherlands), a heparinoid with a mean molecular weight of 5500 d, is obtained from the intestinal mucosa of the pig after removal of heparin. It is a mixture of sulfated glycosaminoglycans with low molecular weight: heparan sulfate (84%), dermatan sulfate (12%), and chondroitin sulfate (4%) [1, 2]. Only a subfraction (4%) of heparan sulfate contains the pentasaccharide sequence, common to heparin and to low-molecular-weight heparins, that has a high affinity to antithrombin III. This subfraction acts through the selective inhibition of factor Xa through antithrombin III, which leads to the inhibition of thrombin generation. The fraction of heparan sulfate with a low affinity for antithrombin III does not affect coagulation factors Xa and IIa but contributes substantially to antithrombotic activity, probably through an endothelial cellular mechanism [3]. The dermatan sulfate component of danaparoid activates heparin cofactor II, which acts at the level of factor IIa. The synergistic activity of these three components determines the antithrombotic profile. As reflected in its anti-factor Xa:anti-factor IIa inhibitory ratio of more than 28:1, danaparoid is a more selective inhibitor of factor Xa than heparin or the low-molecular-weight heparins. The dose-related response to danaparoid remains gradual and linear over a wide dosing range, which may contribute to its safety as an antithrombotic drug. Compared with heparin and low-molecular-weight heparins, danaparoid has almost no effect on physiologic platelet function and has low cross-reactivity with heparin-induced antibodies against platelets. The wide therapeutic range of danaparoid and its minimal effect on platelets may render it a safer anticoagulant than heparin or low-molecular-weight heparins. Treatment with unfractionated heparin is limited by the drugs pharmacokinetic, biophysical, and antihemostatic (nonanticoagulant) properties. Heparin must be given in sufficient quantities under frequent monitoring, its dose-response curve is nonlinear and unpredictable in individual persons, and the risk for bleeding increases with increasing doses and duration of treatment. Danaparoid has been shown in animal studies to be more effective than standard heparin or two different low-molecular-weight heparin preparations in preventing the extension of experimentally induced venous thrombi [4]. It has been both safe and effective in the prophylaxis of deep venous thrombosis in patients having cancer surgery [5], hip-fracture surgery [6], or hip-replacement surgery [7] and in patients with nonhemorrhagic stroke [8]. It has been used as an anticoagulant during hemodialysis [9, 10] and in patients with heparin-induced thrombocytopenia [11, 12] or disseminated intravascular coagulation [13]. Data from studies of the treatment of deep venous thrombosis in patients with hemorrhagic stroke indicate that treatment with danaparoid can prevent the extension of venous thromboembolism without aggravating cerebral bleeding [14]. No study has yet assessed the efficacy and safety of danaparoid in the treatment of patients presenting with acute deep venous thrombosis or pulmonary embolism. Danaparoid has a bioavailability of 100% after subcutaneous administration; the bioavailability of unfractionated heparin after subcutaneous injection is only 20% to 30%. Therefore, danaparoid is particularly suitable for subcutaneous administration, much like the low-molecular-weight heparin preparations [15-19], which have a bioavailablity of approximately 90%. Our study was designed to assess the efficacy and safety of two doses of subcutaneously administered danaparoid and of continuous intravenous administration of unfractionated heparin as initial treatment in patients presenting with acute proximal deep venous thrombosis of the leg, pulmonary embolism, or both. Methods Study Design Our study was a randomized, open, parallel-group clinical trial done in one university hospital and two university-affiliated hospitals in the Netherlands. The study protocol and forms giving informed consent were approved by the institutional review board at each hospital. Patients All patients gave witnessed informed consent before being entered into the study. Men and women 18 years of age or older who presented with clinical symptoms of acute proximal deep venous thrombosis of the leg or pulmonary embolism of no more than 7 days duration were eligible. Patients were excluded if they had had intracranial bleeding within 2 months or resuscitation by external chest compression within 48 hours; if they were allergic to heparin; if they were pregnant; if they were receiving treatment with coumarin derivatives; if they had been treated with thrombolytic drugs within 7 days; or if they were receiving ongoing treatment with aspirin, nonsteroidal anti-inflammatory drugs, dextran, or fibrinolytic drugs. The provisional diagnosis of venous thrombosis or pulmonary embolism had to be confirmed within 48 hours after the start of study treatment by compression ultrasonography or contrast venography (whichever could be done soonest) or by ventilation-perfusion lung scan. Treatment was discontinued and the patient was excluded from the study if the clinical diagnosis was not confirmed. Enrollment began in March 1991 and continued through August 1992. Dosing Schedule The efficacy and safety of two dosing schedules of danaparoid were compared with the efficacy and safety of continuous intravenous unfractionated heparin. The schedule for low-dose danaparoid was 1250 anti-factor Xa units given as an intravenous bolus, followed by subcutaneous doses of 1250 anti-factor Xa units every 12 hours. The schedule for high-dose danaparoid was 2000 anti-factor Xa units given as an intravenous bolus, followed by subcutaneous doses of 2000 anti-factor Xa units every 12 hours. The first subcutaneous injection was simultaneous with the intravenous bolus injection. The second subcutaneous injection was given at the time of the first of the routine twice-daily injections, unless this was within 6 hours of the first injection. Unfractionated heparin was given intravenously as a loading dose of 2500 U and was followed by an initial maintenance dose of 30 000 U every 24 hours. This maintenance dose was adjusted to reach activated partial thromboplastin 2.5 to 3.5 times the control values; these times were equivalent to a heparin level of 0.25 to 0.40 U/mL. This was measured daily and 4 hours after any dose adjustment. Study treatment was given for at least 5 days and was continued until an international normalized ratio of at least 3.0 was achieved with oral anticoagulation therapy, which was started 48 hours after the initiation of study treatment. The oral anticoagulant dose was calculated daily using the Thrombotest (Nyegaard and Co., Oslo, Norway; international sensitivity index, 0.94); this was done each morning using plasma samples taken before the morning dose had been given. If the international normalized ratio was below the target level after 8 days of study treatment, the attending physician decided whether to continue heparin therapy, continue danaparoid therapy, or switch patients being treated with danaparoid to intravenous heparin. Study treatment was randomized as follows. Consecutively numbered, identical boxes were kept in each hospital pharmacy; each box contained one of the three treatments, randomized per hospital. After giving informed consent, a patient was treated with medication from the next consecutive box. The investigators were blinded to the randomization schedule. Only when the study medication for one individual patient was delivered did the treatment become known. After being assigned to treatment, patients were excluded from the efficacy analysis only if diagnosis of deep venous thrombosis or pulmonary embolism could not be confirmed within 48 hours of admission. Evaluations and Scheduling The primary method of assessment for recurrence or extension was repeated ultrasonography of the leg, contrast venography, ventilation-perfusion scanning, or both contrast venography and ventilation-perfusion scanning. Assessment was done after at least 5 days and at most 8 days of study treatment, within 24 hours after stopping treatment, or if clinically indicated. Institutional physicians, who were blinded to treatment assignments, interpreted venograms, ultrasonograms, and lung scans. Clinical evidence of recurrence or extension was defined as documented clinical circumstances suggestive of venous thromboembolic disease leading to the discontinuation of study treatment. A daily physical examination (including tests for hemoglobin level, platelet count, and leukocyte count) and a urinalysis to test for erythrocyte count were done. Liver function tests were done and creatinine levels were measured before and at the end of study treatment. Plasma used to measure amidolytic anti-factor Xa activity was collected at the time of screening and at treatment days 2 and 4 (before and 2.5 hours after the morning injection on both days). This plasma was frozen at 20C and stored until assay. Plasma samples were collected at the time of screening for determination of activated partial thromboplastin times before study treatment. Follow-up assessment was done 2 months after the initiation of study treatment to gather information on state of health, recurrence or extension of venous thromboembolism, and bleeding complications. Compression Ultrasonography To establish the extent of thrombosis using ultrasonography [20], the deep venous system was divided into six segments: lower popliteal, upper popliteal, inferior femoral, mid-femoral, upper femoral, and common femoral veins. Each patient was first examined in the supine position so that the superficial femoral, common femoral, and iliac vein segments could be assessed. Patients were then examined in the prone position so

Factor V Leiden in central venous catheter-associated thrombosis

Summary. 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.

Effects of low doses of aspirin, 10 mg and 30 mg daily, on bleeding time, thromboxane production and 6-keto-PGF1α excretion in healthy subjects

To compare the long term effects of two low doses of aspirin taken daily, we performed a placebo-controlled cross-over study in 19 normal subjects. Aspirin 10 mg daily for 3 weeks caused a significant inhibition by 61 +/- 12 percent of platelet thromboxane B2 generation but had no effect on the Simplate bleeding time. Aspirin 30 mg daily for 3 weeks reduced thromboxane B2 production by 94 +/- 5 percent and caused a significant prolongation of the bleeding time, 1.6 times the control value. No cumulative inhibitory effects were observed after the first week of treatment. Both doses did not effect the urinary excretion of 6-keto-PGF1 alpha.

Low-Density Lipoprotein Enhances Platelet Secretion Via Integrin-αIIbβ3–Mediated Signaling

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 14C-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 αIIbβ3, a polyclonal antibody against αIIb, and in the presence of the fibrinogen peptides GRGDS and HHLGGAKQAGDV; (2) in αIIbβ3-deficient platelets; and (3) after dissociation of αIIbβ3. In contrast, binding of 125I-LDL to normal platelets in the presence of PAC1, anti-αIIb, GRGDS, and HHLGGAKQAGDV, and to αIIbβ3-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 αIIbβ3 after platelet-LDL interaction. Platelets deficient in fibrinogen (<0.5% of normal) or von Willebrand Factor (<1% of normal) but containing normal amounts of other ligands for αIIbβ3 preserved responsiveness to LDL, indicating that occupancy of αIIbβ3 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-αIIbβ3.

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.

ATP‐ADP compartmentation in storage pool deficient platelets: correlation between granule‐bound ADP and the bleeding time

Platelets contain two major compartments of ATP and ADP, the cytosol and the dense granules. We separated the two compartments by controlled digitonin‐induced cell lysis and measured both fractions directly in the platelets of 16 patients with storage pool deficiency. The total contents of ATP and ADP in the platelets of these patients was significantly lower than in normal controls. This was primarily caused by decreased amounts of ATP and ADP in the granule compartment, but also cytosolic ADP was low in these patients. In contrast, cytosolic ATP was not significantly decreased. The lower the amount of granule‐bound ATP and ADP, the longer was the bleeding time in these patients. The best correlation was found with granule ADP. These data may indicate that ADP stored in platelet dense granules plays a role in the arrest of bleeding.

Biochemical and immunohistochemical characteristics of CD62 and CD63 monoclonal antibodies. Expression of GMP-140 and LIMP-CD63 (CD63 antigen) in human lymphoid tissues.

SummaryDuring 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.

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.