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We reported previously that two otherwise identical training programs at lower (LI) and higher intensity (HI) similarly reduced resting systolic blood pressure (BP) by approximately 4-6 mmHg. Here, we determined the effects of both programs on BP-regulating mechanisms, on biomarkers of systemic inflammation and prothrombotic state and on the heart. In this cross-over study (3 × 10 weeks), healthy participants exercised three times 1 h/week at, respectively, 33% and 66% of the heart rate (HR) reserve, in a random order, with a sedentary period in between. Measurements, performed at baseline and at the end of each period, involved blood sampling, HR variability, systolic BP variability (SBPV) and cardiac magnetic resonance imaging. Thirty-nine participants (18 men; mean age 59 years) completed the study. Responses were not different between both programs (P>0.05). Pooled data from LI and HI showed a reduction in HR (-4.3 ± 8.1%) and an increase in stroke volume (+11 ± 23.1%). No significant effect was seen on SBPV, plasma renin activity, basal nitric oxide and left ventricular mass. Our results suggest that the BP reduction observed appears to be due to a decrease in systemic vascular resistance; training intensity does not significantly affect the results on mechanisms, biomarkers and the heart.

It is not well known which exercise intensity is needed to obtain blood pressure reductions in response to endurance training. We therefore compared the effect of training at lower and higher intensity on blood pressure, and, in addition on other cardiovascular risk factors, in at least 55-year-old sedentary men and women.We used a randomized crossover design comprising three 10-week periods. In the first and third periods, participants exercised at, respectively, lower and higher intensity (33 and 66% of heart rate reserve) in random order, with a sedentary period in between. Training programmes were identical except for intensity and were performed three times, 1 h per week. Thirty-nine (18 men) out of 48 randomized participants completed the study; age averaged 59 years.The change of aerobic power from baseline to the end of each period was more pronounced (P0.05) with higher intensity (+3.70 ml/kg min; P0.001) than with lower intensity training (+2.31 ml/kg min; P0.001). Systolic blood pressures at rest and during submaximal exercise were reduced with both intensities (P0.01), whereas diastolic office blood pressure was significantly reduced after higher intensity only (P0.01). There were no significant differences in blood pressure reduction between intensities. Ambulatory blood pressure remained unchanged after training. Only higher intensity training reduced weight (-1.09 kg; P0.001), body fat (-0.85%; P0.001), plasma triglycerides (-0.17 mmol/l; P0.05) and oxidized low-density lipoprotein (-5.92 U/l; P0.01).Higher and lower intensity training reduces systolic office and exercise blood pressure to a similar extent, but does not alter ambulatory blood pressure; only higher intensity training favourably affects anthropometric characteristics and blood lipids.

Udgivelsesdato: 2008-May OBJECTIVE: Most intensive care deaths beyond the first few days of critical illness are attributable to nonresolving organ failure, either due to or coinciding with sepsis. One of the mechanisms that is thought to contribute to the pathogenesis of organ failure is microvascular thrombosis. Recently, we reported significant improved survival and prevention of organ failure with the use of intensive insulin therapy to maintain normoglycemia for at least several days. We hypothesize that intensive insulin therapy also prevents severe coagulation abnormalities thereby contributing to less organ failure and better survival. DESIGN: This was a preplanned subanalysis of a large randomized controlled study, conducted at a university hospital medical intensive care unit. The intervention was strict blood glucose control to normoglycemia with insulin. RESULTS: Mortality of intensive insulin-treated patients was lower than that of conventionally treated patients for all classes of upon-admission disseminated intravascular coagulation (DIC) scores, except for those patients with overt DIC scores of 6 or higher (for DIC < 5, p = 0.003; for DIC > or = 5, p = 0.4). There was no effect of insulin therapy on any of the fibrinolytic, coagulation, or inflammatory parameters tested. CONCLUSIONS: This negative observation does not support a key role for these systems in explaining the clinical benefit of intensive insulin therapy, although a short-lived effect within 5 days of treatment cannot be excluded.

El pdf del artículo es la versión post-print.-- et al., The role of Gas6 in endothelial cell (EC) function remains incompletely characterized. Here we report that Gas6 amplifies EC activation in response to inflammatory stimuli in vitro. In vivo, Gas6 promotes and accelerates the sequestration of circulating platelets and leukocytes on activated endothelium as well as the formation and endothelial sequestration of circulating platelet-leukocyte conjugates. In addition, Gas6 promotes leukocyte extravasation, inflammation, and thrombosis in mouse models of inflammation (endotoxinemia, vasculitis, heart transplantation). Thus, Gas6 amplifies EC activation, thereby playing a key role in enhancing the interactions between ECs, platelets, and leukocytes during inflammation., This work was supported in part by the Swedish Research Council (07 143), by the Spanish Ministry of Science and Technology (grants SAF2004-07 539-C02 and BES2003-0230), by a grant from the Swiss National Foundation for Scientific research (PPOOB-106690/1), by a grant from the Belgian National Fund for Scientific Research in Flanders (FWO; G0265.01), by grant GOA2004/09 from the Concerted Research Activities, Belgium, and by an unrestricted Bristol-Myers-Squibb grant (P.C.). E.L. is a postdoctoral fellow of the Dr E. Dekker Program of the Dutch Heart Foundation (2000T041), M.T. is a postdoctoral research fellow of the Belgian Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT) and FWO.

Between January 2004 and December 2005, out of 14 patients with decompensated heart failure who were treated with an INCOR left ventricular assist device (Berlin Heart AG, Berlin, Germany), 10 patients were kept on a long-term regime of low molecular weight heparin (LMWH) and antiplatelet therapy. The treatment objective was bridge-to-transplantation. All patients received LMWH in therapeutic doses according to body weight, in combination with daily aspirin 160 mg, clopidogrel 75 mg, and three times dipyridamole 75 mg. Effectiveness of the low molecular weight regime was monitored through measurement of antifactor Xa activity (base and peak levels). Antiplatelet therapy was monitored through weekly platelet function tests. Within this group of 10 patients, six patients successfully received transplants and four patients died, the latest death after 405 days of INCOR support. Causes of death were sepsis, intestinal hemorrhage, acute right ventricular failure, and one major stroke. Long-term management of INCOR assist devices using a combination of LMWH and antiplatelet therapy is feasible. This treatment strategy can serve as an alternative to oral anticoagulants.

Many investigators have been intrigued by the paradoxical association of a circulating anticoagulant, first called lupus anticoagulant by Feinstein and Rapaport [1], with a tendency to develop thrombosis, as initially described by Walter Bowie [2]. Work in Leuven on this topic started when Luis Carreras, an Argentinian hematologist, joined the laboratory of blood coagulation at this university in 1979. At that time, the head of the laboratory was Marc Verstraete. Luis had a particular interest in antibody-mediated coagulation disorders, and had prepared reviews on thrombosis and thrombocytopenia induced by heparin [3] and on the lupus inhibitor [4]. In Leuven, he joined Jos Vermylen, senior member of the laboratory, and an internist with particular interest in hemostasis, thrombosis and vascular disease. As such, Professor Vermylen was involved in both laboratory research and patient care.

The association between Hodgkin's lymphoma, antiphospholipid syndrome and severe mitral insufficiency is a very rare event. We report on a 25-year-old female patient suffering from Hodgkin's lymphoma and presenting with thromboembolic events and severe mitral insufficiency. The possible link between these symptoms being antiphospholipid antibodies, is discussed briefly.

The objective was to investigate whether the platelet dysfunction in cardiac surgery is caused by hemodilution or by shear stress due to cardiopulmonary bypass (CPB). Platelet count and function were prospectively analyzed in two groups of patients undergoing cardiac surgery either with or without CPB (n = 40). In the first study (n = 20; 10 patients with and 10 without CPB), platelet counts were assessed at seven time points. In the second study (n = 20; 10 patients with and 10 without CPB), platelet function was studied with platelet aggregometry at different points during surgery: (a) after induction of anesthesia; (b) after sternotomy; and (c) 1 h after heparin. In the first study, the CPB group showed a significant decrease in platelet count starting after sternotomy (230 +/- 34 vs. 182 +/- 25, P < 0.05) and a maximum decrease at day 1 postoperative (96 +/- 34, P < 0.05). A similar observation was made in the non-CBP group. In the second study, a significant decrease of ADP (54 +/- 13% vs. 38 +/- 9%, P < 0.05), AA (76 +/- 16% vs. 22 +/- 14%, P < 0.05), and Collagen (66 +/- 13% vs. 37 +/- 11%, P < 0.05) induced platelet aggregation was observed at MOMENT d compared to the beginning of surgery in the CPB group. In the non-CBP group a significant decrease was observed in AA-induced platelet aggregation at MOMENT d (83% +/- 4 vs. 44% +/- 14, P < 0.05). The reduction in platelet count is similar with or without cardiopulmonary bypass and is due to pure hemodilution. Platelet function reduces significantly after heparin administration. Hemodilution and predominantly heparin are the causes of platelet dysfunction after cardiac surgery.

Objective - In Belgium oral anticoagulation therapy is mainly supervised by general practitioners (GPs).This study aims to evaluate the quality of management of oral anticoagulation by Belgian GPs and to verify the relation between time in range and a set of potentially influencing co-variables. Methods - In a retrospective cross-sectional study, involving 66 GP-practices, the INR-values obtained over a 6-month period were analysed. All INR-values were determined by a single clinical laboratory and additional medical information was provided by the GPs. Linear mixed models have been used to model the patient-specific percentage INR in target as a function of different co-variables. Results - 737 patients were included in the study. Patients who underwent a surgical intervention with an interruption of the anticoagulation during the study were excluded. Patients were only included after the initial starting-up period. 5,890 INR-values were obtained. A total of 92,566 days of therapy was evaluated. 50% of the day values were within 0.5 INR-units from target (and 66% within 0.75 INR-units from target). In a multiple regression model, a significant relation between the percentage of time in range and the target INR (2.5 or 3.5) and the gender of the patient was shown. The incidence rate for major bleeding was 5.5/100 patient years (and 3.5/100 patient years for thrombo-embolic events). Conclusion - The quality of management of oral anticoagulation by the GPs in Belgium is suboptimal. It is unknown whether interventions such as guidelines, feedback, point-of-care monitoring and computer-assisted anticoagulation monitoring could improve the results.

The hemostatic system comprises platelet aggregation, coagulation and fibrinolysis also termed primary, secondary and tertiary hemostasis. From the platelet transcriptome 6000 mRNA species and represent receptors, ion channels, signalling molecules, kinases, phosphatases, and structural, metabolic and regulatory proteins. This abundance of regulatory proteins points towards the importance of signal transduction in platelet function. First platelets adhere to collagen, this induces activation signals such as TXA(2) that induces further Ca(2+) increase. Consecutively, fibrinogen binds to the integrin alpha(IIb)beta(3) resulting in aggregation.This self-amplifying process is controlled by signals, from endothelial cells, to restrict the platelet plug to the site of vessel injury. Secondary hemostasis (coagulation) consists of an extrinsic and intrinsic pathway. Thrombin is generated via Factor Xa resulting from the extrinsic tenase reaction that is turned of by tissue factor pathway inhibitor. While thrombin generation is maintained via positive feedback mechanisms activating factors V, VIII and XI. Excess thrombin is inhibited by antithrombin or by autodownregulation via activation of protein C. Since minor injuries are common, platelets and plasma clotting factors constantly produce clots to stop bleeding. If clots remained after the tissue healing, the vascular bed would become obstructed with clots therefore this is regulated by fibrinolysis, tertiary hemostasis. Tissue-type plasminogen activator synthesised by the endothelium, converts plasminogen to plasmin, the clot lysis enzyme. Plasmin clears the blood vessels by degrading fibrin. Fibrinolysis is controlled by plasminogen activators inhibitor (PAI-1), alpha2-antiplasmin and alpha2-macroglobulin, and thrombin-activatable fibrinolysis inhibitor (TAFI).

Venous thromboembolism(VTE) in patients suffering from the antiphospholipidsyndrome (APS) has been reported in almost any location of the vessel tree and the risk of recurrences has been found in several studies to be more closely associated with the presence of lupus anticoagulant than with the positivity for anti-cardiolipin antibodies. The thrombophilic state of APS raises the problem of the secondary prophylaxis to avoid VTE recurrences. For APS patients with VTE, published data appear to support a longer warfarin treatment if compared with the standard management of anti-phospholipid (aPL)-negative patients with VTE. The question of how long oral anticoagulant treatment should be continued for APS patients, however, remains unanswered. Concerning the intensity of anticoagulation, several authors recommend a target international normalized ratio (INR) between 3.0 and 4.0 to efficiently protect from VTE recurrences.A recentdecisionanalysisstudy does support such a suggestion. On the contrary, in a few prospective studies regimens with lower target INRs appear to be effective, and some authors therefore recommend a target INR of between 2.0 and 3.0. Specific large and prospective trials are needed to address this question. Until such information becomes available, individualized treatment according to the patient’s individual risk factors for both bleeding and thrombosis is the general practice.

SUMMARY Background : Aspirin is widely used as an anti-thrombotic drug; however, it has been suggested that enteric-coated formulations of aspirin may be less bioavailable and less effective as anti-thrombotic agents. Aim : To assess the effect of a formulation of enteric-coated, low-dose (81 mg) aspirin on serum generated thromboxane B2 and platelet aggregation in healthy subjects. Methods : Twenty-four subjects participated in a double-blind, randomized, placebo-controlled, parallel-group, multiple-dose study. Twelve subjects in each of two groups received a daily oral dose of enteric-coated aspirin (81 mg) or matching placebo for 7 days. Serum thromboxane B2 and platelet aggregation (using 1 mm arachidonic acid and 1 µg/mL collagen as agonists) were measured 1–3 days prior to day 1, on day 1 (prior to therapy) and 4 h after the last dose on day 7. Results : After seven daily doses of enteric-coated aspirin, the mean percentage inhibition from baseline of ex vivo generated serum thromboxane B2 was 97.4%, compared with a 7.8% increase after placebo treatment. The mean percentage inhibition of arachidonic acid- and collagen-induced platelet aggregation was 97.9% and 70.9%, respectively, following enteric-coated aspirin, compared with − 1.0% and 2.7%, respectively, after placebo. Conclusions : The anti-platelet effects of multiple, daily, low-dose aspirin (as assessed by inhibition of serum thromboxane B2 and platelet aggregation) are not adversely affected by enteric coating.

Two siblings, a boy age 12 and his sister age 4 years, presented with proteinuria and hematuria, hypertension, and chronic hemolytic anemia. At age 13 years, the boy developed an episode of severe hypertensive encephalopathy and transient renal failure. Both children are attending normal school, have no neurologic symptoms, and only minimal pigmentary retinal abnormalities. Renal biopsy showed a chronic thrombotic microangiopathic nephropathy. Both patients had hyperhomocysteinemia and mild methylmalonic aciduria. Fibroblasts showed decreased cobalamin uptake, reduced methyl- and adenosyl-cobalamin formation, and deficient incorporation of formate and propionate, compatible with the Cbl-C complementation group, but milder than that found in cells from most patients. Both patients and their father carry a balanced reciprocal translocation. Parenteral hydroxycobalamin treatment reduced the homocysteine levels, and methylmalonic acid disappeared. Increasing the dosage of hydroxycobalamin from 1 to 2.5, then 5 mg daily together with betaine, further reduced homocysteine levels (boy from 118 to 23 microM and girl from 59 to 14 microM). With this treatment, hemolysis has stopped, hematuria has disappeared, proteinuria has almost normalized, and creatinine clearance has been stable. Investigations for chronic thrombotic microangiopathy should include testing for this unusual but treatable disorder, regardless of age of presentation.

Return of blood activated by tissue factor is the main culprit for triggering the coagulation cascade. When this activated blood is diverted from the cardiopulmonary bypass (CPB) circuit, it becomes possible to evaluate the effect of surface treatment on platelet and complement activation. Twenty adult patients undergoing elective coronary artery bypass grafting (CABG) were randomly assigned either to a control group ( n= 10) or to a group in which the CPB circuit was completely coated with phosphorylcholine ( n= 10). Plasma concentrations of platelet factor 4 (PF4), β-thromboglobulin (βTG), C3, C3d, C4, TCC, thrombin generation, haptoglobin and free haemoglobin, as well as blood loss, were measured. No significant differences between the two groups were found for haemolysis and thrombin generation. The mean total release of PF4 and βTG during CPB was 9338± 17303 IU/ml/CPB and 3790± 4104 IU/ml/CPB in the coated group versus 22192± 13931 IU/ml/CPB ( p= 0.011) and 8040± 3986 IU/ml/CPB ( p= 0.005) in the control group. Blood loss was 30% less in the coated group compared to the control group. Phosphorylcholine coating appears to have a favourable effect on blood platelets, which is most obvious after studying the changes during CPB. Clinically, this effect resulted in a 30% reduction in blood loss.

SummaryAlternatively spliced GNAS1 and XL-GNAS1, encoding respectively the stimulatory G-protein α-subunit (Gsα) and the extra-large stimulatory G-protein α-subunit (XLsα), are located on the imprinted chromosomal region 20q13.12-13. We presently report a functional polymorphism in the imprinted XL-GNAS1 gene. In three patients, a 36 bp insertion and two basepair substitutions flanking this insertion were found in the paternally inherited XL-GNAS1 exon 1. They clinically manifest an enhanced trauma-related bleeding tendency and a variable degree of mental retardation. A platelet aggregation inhibition test to evaluate Gs function was developed. Their platelets display Gs hyperfunction and an enhanced cAMP generation upon stimulation of Gs-coupled receptors. The prevalence of the XLsα insertion in a normal control group was 2.2%. Normal controls, inheriting the insertion maternally, had a normal platelet Gs activity, whereas controls inheriting the insertion paternally had increased inducible platelet Gs activity, defining the insertion as a functional polymorphism. This paternally inherited XLsα insertion represents a new genetic cause of an inherited bleeding tendency, although to a variable degree.

SummaryQuantification of lupus anticoagulant (LA) in clinical samples is hampered by the lack of a suitable standard of activity. We evaluated the use of mAbs displaying LA activity for this purpose. As most patient samples contain both β2Glycoprotein I (β2GP1) and prothrombin dependent LA, a combination of two mAbs, one of each specificity, was added to normal plasma in a concentration from 0 to 60 g/ml. Eight assay systems using different reagents and instruments were used. The calibration curves were linear for all but one, with marked differences between the responsiveness to each mAb. A panel of plasmas from 69 patients with persistent LA diagnosed using the SSCISTH criteria was tested. An antiphospholipid syndrome (APS) was present in 40, whereas 29 were asymptomatic. LA activities of individual plasmas varied between assays (p

Activation of both coagulation and fibrinolysis in childhood hemolytic uremic syndrome.BackgroundThrombotic microangiopathy is the fundamental lesion in diarrhea-associated hemolytic uremic syndrome. The extent of the lesion in the renal parenchyma determines the severity and outcome of the disorder, bilateral renal cortical necrosis being the worst end of the spectrum. In the early years, intravascular coagulation was considered the most important pathogenic mechanism. Yet, individual coagulation factors were normal in the vast majority of patients and therapy with anticoagulants did not alter the course. Recent studies indicate that impaired fibrinolysis might be of importance.MethodsWe studied seven variables of the coagulation pathway (PT, aPTT, fibrinogen, FVIII:c, von Willebrand factor, thrombin-antithrombin complexes, prothrombin fragments 1+2) and seven parameters of the fibrinolytic system (plasminogen, alpha2-antiplasmin, C1-esterase inhibitor, tissue-type plasminogen activator, urokinase-type plasminogen activator, plasminogen activator inhibitor type 1, D-dimer) in 24 pediatric patients with diarrhea-associated hemolytic uremic syndrome and in 15 children with acute renal failure not due to hemolytic uremic syndrome. Samples were collected at diagnosis and every second day thereafter for a period of ten days. Additional samples were collected from patients who underwent dialysis, that is, before and after each session from those subjected to hemodialysis and every day from those subjected to peritoneal dialysis. The obtained data were compared with data from a control group consisting of healthy children.ResultsOur data show four important features. (1) A significant increase in both thrombin-antithrombin complexes (P < 0.005) and prothrombin fragments 1 + 2 (P < 0.001) is observed in hemolytic uremic patients as compared to patients with acute renal failure of other causes. This finding is clearly indicative for an activation of the coagulation pathway. (2) Patients with the hemolytic uremic syndrome have significantly higher D-dimer levels, a sensitive marker of fibrin-specific fibrinolysis, as compared to patients with acute renal failure of other causes (P < 0.005). (3) Levels of plasminogen activator inhibitor-1 (active antigen as well as plasminogen activator inhibitor-1 activity) are not different in both patient groups. In contrast, plasma levels of tissue-type plasminogen activator and urokinase-type plasminogen activator are significantly higher in the hemolytic uremic patients than in those with acute renal failure of other causes (P < 0.01 and P < 0.05 respectively). (4) Hemodialysis leads to an increase in tissue-type plasminogen activator antigen and a decrease of plasminogen activator inhibitor-1 activity levels.ConclusionsOur data demonstrate that in children with diarrhea-associated hemolytic uremic syndrome, limited intravascular coagulation occurs, without evidence of impaired fibrinolysis.

There is accumulating evidence showing that lupus anticoagulants (LA) are more strongly associated with thrombosis than anticardiolipin antibodies. In addition, indirect evidence has been presented indicating that beta2GPI-dependent LA are more strongly associated with thrombosis than prothrombin-dependent LA. From this, one may assume that anti-beta2GPI antibodies with LA activity are more pathogenic than anti-beta2GPI antibodies without LA activity. Therefore, it is of the utmost importance to understand the molecular basis on which some anti-beta2GPI antibodies behave as LA. In this presentation, the current knowledge on the interaction of beta2GPI with phospholipids and with anti-beta2GPI antibodies is reviewed and an integrated model for the anti-beta2GPI-dependent LA activity is proposed with implications for a pathogenic role of these particular antibodies.

SummaryThe laboratory diagnosis of lupus anticoagulants (LA) remains difficult despite internationally accepted guidelines for its detection. Several interlaboratory surveys have shown poor agreement between laboratories. Further standardization of LA testing will to a large extent depend on better insights on the mechanisms by which LA affect phospholipid-dependent coagulation assays as well as on the availability of well characterized and internationally accepted reference materials and control specimens. We recently raised a series of murine monoclonal antibodies against human β2GPI (anti-β2GPI moabs), a phospholipid-binding protein directly involved in the interaction between certain lupus anticoagulants and phospholipids. In this study we report on the use of LA positive anti-β2GPI moabs as easy to handle reference and control material. The relative LA responsiveness of various phospholipid-dependent clotting assays was determined on plasmas spiked with such moabs and compared well with that determined on LA positive plasma samples. Plasmas spiked with LA positive anti-β2GPI moabs were also used as control specimens to study the interlaboratory precision of LA testing. With these control specimens, low interassay coefficients of variation were obtained. Our results indicate that LA positive anti-β2GPI moabs have potential for the production of control specimens that could be made available to routine hemostasis laboratories to assess intra-laboratory precision of LA testing and to manufacturers to control batch-to-batch variability of their reagents.

Two case reports of pregnant patients with antiphospholipid antibodies and HELLP syndrome (hemolysis, elevated liver enzymes and low platelets) are presented. Attention is mainly drawn to the hepatic necrosis and the underlying pathophysiology.

SummaryAntibodies to factor VIII (inhibitors) are usually produced at the beginning of treatment with factor VIII and are rare in multitransfused patients. Such antibodies are deemed to be patient-related, as supported by the description of a number of associated risk factors. However, a second category of inhibitors has recently been identified, namely antibodies occurring in multitransfused patients as a result of exposure to a particular factor VIII concentrate. A first outbreak of product-related inhibitors was recently described. The present paper describes the second well-documented occurrence of such inhibitors.Eight out of 140 multitransfused patients with severe haemophilia A developed an inhibitor to factor VIII shortly after changing treatment to a double-virus inactivated plasma-derived factor VIII concentrate. In addition to solvent-detergent treatment, this concentrate was pasteurised at 63° C for 10 hours. Exposure to the pasteurised product before inhibitor detection ranged from 9 to 45 days. Inhibitor titers varied between 2.2 and 60 Bethesda Units and recovery of transfused factor VIII ranged from 0.21 to 0.68 (expressed as IU/dl factor VIII rise per IU/kg administered). In contrast to usual inhibitors in haemophilia A patients, these product-related inhibitors showed complex inhibition kinetics. They were found specific for the factor VIII light chain. The inhibitors gradually declined when exposure to the pasteurised product was stopped, despite further treatment with other factor VIII concentrates. The present data stress the importance of carefully monitored clinical studies, both in previously treated and previously untreated patients, before introduction of a new or modified clotting factor concentrate.

In the perioperative period, artificial colloids are most often infused in doses of 500-1000 mL intravenously. This randomized study compared the effects on intravascular volume and coagulation of approximately 2000 mL of two isooncotic artificial colloids: 6% hydroxyethyl starch (HES; MW 200,000; substitution ratio 0.40-0.55) and 3% modified fluid gelatin (GEL). We hypothesized more pronounced hypocoagulation with HES and a weaker intravascular volume effect of GEL. Forty-two patients, scheduled for primary total hip replacement, were allocated randomly to receive HES or GEL during acute normovolemic hemodilution and subsequent further intraoperative hemodilution. Blood samples were taken before and after 500 mL and 1000 mL of acute normovolemic hemodilution; intraoperatively after 20 mL/kg of artificial colloid and at the end of colloid infusion; on arrival in the recovery room; and 3 h later. We quantified: 1) coagulation variables; 2) blood loss; 3) hemodynamic stability; 4) necessary infusion volume; 5) interstitial extravasation, calculated from plasma volunteers measured using albumin marked with technetium-99m and iodine-125, respectively; 6) percentage volume effect at the end of the study as well as hematocrit, total serum protein, and colloid osmotic pressure. Intraoperative volume therapy was guided by radial systolic pressure and systolic pressure variation, mixed venous hemoglobin saturation in the pulmonary artery, and pulmonary capillary occlusion pressure.(ABSTRACT TRUNCATED AT 250 WORDS)

During the last decade a new clinical syndrome, the antiphospholipid syndrome, was described. This syndrome, which gradually developed over many years, is now defined as the association of so-called antiphospholipid antibodies with arterial or venous thrombosis, recurrent fetal loss, thrombocytopenia, or neurologic disorders. Antiphospholipid antibodies, namely lupus anticoagulants and anticardiolipin antibodies, are closely related phospholipid-binding autoantibodies which are, respectively, measured via their capacity to prolong phospholipid-dependent coagulation assays or via their capability to bind to cardiolipin, a negatively charged phospholipid, immobilized on a microtiter plate. Antiphospholipid antibodies are found in about one third of patients with systemic lupus erythematosus (SLE) or related autoimmune disorders, and their presence concurs with an increased risk for the above-mentioned clinical features. About half of the patients with an antiphospholipid syndrome do not fulfill the criteria...

RGS18 was originally identified as a R4 subfamily member of regulators of G-protein signaling (RGS) with specific expression in hematopoietic progenitors, myeloerythroid cells, and megakaryocytes, though its physiological role in hematopoiesis remained unknown. Here, we show that lentiviral RGS18 overexpression during differentiation of mouse Sca1(+) hematopoietic stem cells induced a 50% increase of megakaryocyte proliferation. RGS18 depletion in zebrafish results in thrombocytopenia, as 66 to 88% of the embryos lack thrombocytes after injection of an ATG or splice-blocking morpholino, respectively. These embryos have no defects in early hematopoiesis, erythropoiesis, or leukocyte number and migration. In addition, all RGS18 depleted embryos have curly tails and an almost absent response to acoustic stimuli. In situ hybridization in zebrafish, Xenopus, and mouse embryos shows RGS18 expression in thrombocytes and/or hematological tissues but also in brain and otic vesicles. RGS18 interferes with development of cilia in hair cells of the inner ear and neuromast cells. On the basis of literature evidence that RGS-R4 members interact with the G-protein-modulated Wnt/calcium pathway, Wnt5b- but not Wnt5a-depleted embryos phenocopy all RGS18 knockdown effects. In summary, our study is the first to show that RGS18 regulates megakaryopoiesis but also reveals its unexpected role in ciliogenesis, at least in lower vertebrates, via interference with Wnt signaling.

Ten healthy male volunteers not previously exposed to hirudin participated in an open trial to evaluate the effects on platelet function and coagulation of recombinant hirudin (rec-hirudin) CGP 39393 1 mg/kg administered intravenously as a bolus injection.

SummaryA patient with a history of habitual abortion, deep venous thrombosis, thrombocytopenia, high titer IgG anticardiolipin antibodies and a clearly positive lupus anticoagulant, was treated during her seventh pregnancy with high-dose intravenous immunoglobulins (IVIg) from the third month onwards. Every month, a daily infusion of 400 mg immunoglobulins per kg body weight was given during five consecutive days. The patient’s pregnancy ended preterm with a live birth, delivered by caesarian section because of a placental abruption. The 1070 g (P20-P25) weighing girl was in good health, apart from a bradycardia, due to dysfunction of the atrioventricular conduction.Each treatment with IVIg resulted in a slight reduction of both anticardiolipin antibodies and lupus anticoagulant levels and in an increase in platelet count. During the six-month observation period, a gradual decline in antiphospholipid antibodies and an increase in platelet count was found. The potential role of anti-idiotypic antibodies, present in the IVIg used for treatment, was studied. In vitro, IVIg were able to reduce the binding of the patient’s anticardiolipin antibodies to cardiolipin coated microtiter plates. The presence of anti-idiotypic antibodies in IVIg was further documented by affinity chromatography and by realtime biospecific interaction analysis (BIA) on a BIA-core instrument. Affinity purified anticardiolipin antibodies were retarded on a column of insolubilized IVIg and a weak interaction was found between IVIg and affinity purified patient antiphospholipid antibodies, coupled to the BIA-core biosensor. In addition, the same technology revealed increased levels of anti-antiphospholipid antibodies in the patient’s plasma following IVIg therapy. The partial and transient reduction of anti-phospholipid antibody levels observed immediately following each treatment course seems compatible with low affinity complexation of idiotype-antiidiotypes, resulting in an accelerated clearance of the autoantibodies. Despite the low affinity for the patient’s idiotypes, the beneficial long term effects observed could be related to an immune regulatory role of these anti-idiotypic antibodies on the synthesis of antiphospholipid antibodies.

BACKGROUND Fibrinogen-dependent cross-linking of glycoprotein (GP) IIb/IIIa on activated platelets is the final mechanism leading to platelet aggregation. Inhibition of this mechanism may result in a novel antithrombotic agent. We studied the activity of MK-383 (L-700,462), a new, nonpeptide GPIIb/IIIa antagonist, in vitro and in vivo, in man. METHODS AND RESULTS MK-383, a nonpeptide tyrosine derivative, dose-dependently inhibited fibrinogen-dependent platelet aggregation, in vitro. Binding of 125I-labeled fibrinogen to activated platelets was prevented in a competitive manner with an IC50 of 10 +/- 4.2 nmol/L. The activity and tolerability of MK-383 were evaluated in a two-part double-blind, placebo-controlled, dose-escalation study in healthy male subjects using 1- and 4-hour intravenous infusions. Effects on ADP- and collagen-induced ex vivo platelet aggregation (APA or CPA) and template bleeding time (TBT) were evaluated. Twenty-four subjects participated in the 1-hour part. Six received placebo and 18 MK-383 in doses ranging from 0.05 to 0.40 microgram.kg-1 x min-1. MK-383 inhibited platelet aggregation and prolonged bleeding time in a dose-dependent manner. APA and CPA were totally inhibited at the end of infusion of 0.4 microgram.kg-1 x min-1 and returned to 55% and 89% of baseline, respectively, at 3 hours after infusion. TBT was prolonged at this dose from 5.0 +/- 1.3 minutes predose to 22.7 +/- 6 minutes at the end of the infusion (P < .01) and was normalized by 3 hours after infusion. In the 4-hour infusion part, 15 subjects received MK-383 (0.1 to 0.2 microgram.kg-1 x min-1), and five received placebo. Complete inhibition of ex vivo platelet aggregation was seen at 0.15 and 0.2 microgram.kg-1 x min-1. At 0.2 microgram.kg-1 x min-1, TBT was prolonged from 4.4 +/- 1.2 to 23.9 +/- 4.3 minutes at the end of infusion (P < .01) and remained slightly prolonged 3 hours after infusion (7.2 +/- 1.8 minutes). No adverse effects were observed in any of the 33 subjects receiving MK-383. CONCLUSIONS The results from this study indicate that MK-383 appears to be well tolerated and active in man. It is the first nonpeptide GPIIb/IIIa antagonist that can be used to investigate the antithrombotic potential of this new class of antiplatelet agents.

SUMMARY BACKGROUND: Anticardiolipin (aCL) and anti-beta(2)glycoprotein I (abeta(2)GPI) antibodies are part of the criteria for antiphospholipid syndrome (APS). Therefore they are widely measured and about 30 commercial kits are available. OBJECTIVES: To investigate the analytical and clinical performances of four fully automated, chemiluminescent assays: HemosIL AcuStar aCL IgG, HemosIL AcuStar aCL IgM, HemosIL AcuStar abeta(2)GPI IgG, and HemosIL AcuStar abeta(2)GPI IgM. METHODS: Cut-off values were assessed by testing 250 blood donors. Total coefficients of variation (CV) were determined with six plasma pools and two controls ranging from 4.3 to 2694 U mL(-1) depending on the assay. Samples from 218 well-characterized patients and 103 controls were measured in three laboratories to determine inter-laboratory variation. The results of the 321 samples were compared with three commercial assays (REAADS, INOVA and VARELISA). RESULTS: Cut-off values were assigned to 20 arbitrary units for all the tests. Total CV ranged from 4.3 to 11.2%. No interference of hemoglobin, bilirubin, triglycerides, heparins and rheumatoid factor was observed. Inter-laboratory variability was low and no sample changed status. Overall status agreement between HemosIL assays and the comparator kits ranged from 82 to 96%. Sensitivity, specificity, agreement when predicting APS and the odds ratios when predicting a thrombotic or obstetric event gave comparable results between HemosIL AcuStar and the three other assays. CONCLUSIONS: Our study demonstrates that the fully automated HemosIL AcuStar aPL assay panel showed similar performances to the three commercial ELISAs commonly used by various laboratories to detect antiphospholipid antibodies.

BACKGROUND Fibrin generation during and after therapy with alteplase may depend on the level of concomitant anticoagulation. The hypothesis that fibrinopeptide A (FPA) levels, as markers of ongoing in vivo fibrin formation, correlate with the angiographic and clinical outcome of thrombolysis is tested. METHODS AND RESULTS Serial plasma FPA levels were determined in 334 patients of the randomized European Cooperative Study Group trial comparing heparin versus placebo plus alteplase and aspirin in patients with acute myocardial infarction. Median FPA levels (with the 10th to 90th percentiles) were 21 ng/ml (2-390 ng/ml) before treatment in placebo-allocated patients (n = 166) and increased to 49 (15-580), 34 (4-320), 27 (2-240), 29 (2-430), and 30 (3-390) ng/ml after 0.75, 3, 12, 24, and 36 hours, respectively. In heparin-allocated patients (n = 168), median baseline FPA values were 18 ng/ml (2-210 ng/ml) and decreased to 6 (1-110), 5 (1-75), 5 (1-60), 7 (1-100), and 10 (1-170) ng/ml at corresponding time points (p less than 0.0001 for the difference at each time point). Adequate anticoagulation, defined as no activated partial thromboplastin time value below twice the pretreatment value at 3, 12, 24, and 36 hours after initiation of treatment, was obtained in 48 patients assigned to heparin. It was associated with normal median FPA levels (less than or equal to 4 ng/ml) at all time points compared with 12 (2-80), 16 (2-240), and 15 (2-240) ng/ml at 12, 24, and 36 hours, respectively, in heparin-assigned but inadequately anticoagulated patients (n = 102, p less than 0.001 for each time point). In the heparin-treated group, median FPA values tended to be lower at all time points in patients with patent vessels than in patients with occluded arteries, but the difference was significant only at 24 hours (p = 0.04). FPA levels did not correlate with clinically apparent recurrent ischemia or with left ventricular thrombosis on two-dimensional echocardiography. CONCLUSIONS During and after thrombolytic therapy with alteplase, the enhanced fibrin generation is suppressed by sustained concomitant anticoagulation with intravenous heparin. Adequate anticoagulation warrants individual titration of the heparin dose. High plasma FPA levels 24 hours after alteplase therapy are specific but insensitive markers of vessel occlusion in anticoagulated patients. They do not correlate with clinical outcome.

The present study compares two new factor VIII preparations currently used in the treatment of haemophilia. A Factor VIII was partially purified from plasma obtained from unpaid voluntary Belgian donors in the blood transfusion centers of Lille and Amsterdam and virus-inactivated by exposure to solvent-detergent (FVIII-SD) or by pasteurization (FVIII-P) respectively. The factor VIII content and the purity of both preparations were assessed in vitro, whereas in vivo we studied the recovery and the plasma half-life of both concentrates. The higher purity of FVIII-SD was confirmed. Factor VIII-P preparations contained more factor VIII than mentioned on the label. Both preparations gave good in vivo recoveries and half-lives. Patients who did not have antibodies to hepatitis B, hepatitis C or HIV at the initial screening, remained negative after six months treatment with the new concentrates. No patients developed neutralizing factor VIII antibodies. Furthermore patients appreciated the ease of administration of both preparations. In conclusion both FVIII concentrates are suited for the treatment of haemophilia A patients.

Two murine monoclonal antibodies, raised against von Willebrand factor (vWF), were used to construct an enzyme-linked immunosorbent assay (ELISA), for quantitation of vWF antigen (vWFAg) in human plasma and platelets. This assay had a lower limit of sensitivity of 0.0001 IU/ml in buffer, and thus is one to two orders of magnitude more sensitive than other ELISA assays which have been reported. The intraassay, interassay and interdilution coefficients of variation were 4.1, 10.4 and 9.9%, respectively. In normal plasma (n = 20), the vWFAg level was 0.83 (range: 0.42–1.25) IU/ml. In normal washed platelets (n = 10), 0.35 (0.25–0.49) IU/109 platelets was found. In plasma obtained from various patient groups the following vWFAg levels (geometric mean and range) were observed: von Willebrand’s disease (n = 19): 0.18 (0.02–0.77) IU/ml; patients with liver cirrhosis (n = 20): 3.73 (1.68–9.20) IU/ml; patients with pregnancy-induced hypertension (n= 20): 4.14 (2.28–7.44) IU/ml and patients with malignant disease (n = 10), 2.54 (1.51–5.60) IU/ml. A linear correlation was found between vWFAg levels measured with a polyclonal antibody based Laurell electroimmunoassay (r = 0.92, n = 58) or with a polyclonal antibody based ELISA (r = 0.94, n = 64). The present assay is based on stable and reproducible reagents and allows the specific measurement of vWFAg in plasma and in platelets. This assay may constitute a useful tool for the further investigation of clinical conditions associated with changes in vWFAg levels. In addition, its high sensitivity may facilitate a more detailed study of platelet vWFAg in normal and in pathological conditions.

International audience; INTRODUCTION: Moderate alcohol consumption is protective against cardiovascular disease (CAD). ADHs are major enzymes of alcohol metabolism. A polymorphism in the alcohol dehydrogenases 1C gene (ADH1C) was reportedly associated with the protective effect of alcohol consumption on CAD risk and risk factor levels. AIMS: The aim of our study was to investigate whether the association of alcohol consumption with metabolic risk factors for CAD is related to ADH1C variants. METHODS: IMMIDIET is a cross-sectional study of 974 healthy male-female pairs living together, randomly recruited in Belgium, Italy and England. The rs698 ADH1C polymorphism was genotyped. A 1-year recall food frequency questionnaire was used to estimate alcohol intake. RESULTS: The intake of alcohol did not vary in relation to ADH1C genotypes. BMI, waist circumference (WC), waist-to-hip ratio, blood pressure, HDL or total cholesterol, triglycerides and FVII:ag levels were positively associated with alcohol intake in men (multivariate ANOVA). Regression coefficient for alcohol and BMI or WC was progressively higher in heterozygotes and gamma 2 homozygotes as compared to gamma 1 homozygotes (p=0.006 and p=0.03 for interaction, respectively). No interaction was found for other risk factors. In women, alcohol intake was positively associated with HDL, LDL and FVII:ag levels but no interaction was found between ADH1C polymorphism and any risk factor. CONCLUSION: Regulation of ADH1C genotype on the association between alcohol consumption, BMI and WC was found in men from different European countries. In men homozygous for the gamma 2 alleles, intake of alcohol was positively associated with both BMI and WC values.

We have investigated the effects of R68070 on platelet function in vitro and in vivo. The drug inhibits U46619induced aggregation (IC, = 1.2 x lo-' mol/L), blocks serum thromboxane formation (IC, = 1 x mol/L), and increases serum prostaglandin (PG)E, and 6-keto-PGF1, levels, indicating that it combines thromboxane receptor blocking and thromboxane synthase inhibiting properties. The thromboxane-dependent aggregation of blood platelets is blocked by R68070, whereas no inhibition of thromboxane indepdndent pathways occurs. A double-blind, randomized, cross-over study was performed on nine volunteers, comparing 400 mg placebo, 400 mg aspirin, and 400 mg R68070. Thromboxane-dependent aggregations ROSTAGLANDINS, formed through the action of P cyclo-oxygenase on arachidonic acid, have profound effects on platelet reactivity.' The intermediary products, cyclic endoperoxides and their metabolite, thromboxane A,, the major product formed in the platelet, act on the same proaggregatory receptor., PGD, and PGI,, the latter formed mainly by endothelial cells but also by white blood cells, inhibit all routes of platelet a~tivation'.~ and local vasoconstriction by virtue of their adenylate cyclase stimulating a~tion.~ In view of these potent actions, a major effort has been undertaken during the last decade to pharmacologically manipulate the formation or activity of prostaglandins. Nonsteroidal, anti-inflammatory drugs inhibit cyclooxygenase and thereby block the formation of all prostaglandins. Aspirin, irreversibly acetylating the enzyme: has been tested exhaustively mainly because platelets are anucleated in contrast to endothelial cells. They are thus unable to quickly recover from an aspirin challenge. This should result finally in a reduction of platelet thromboxane A, (TxA,) formation, while leaving endothelial PGI, formation virtually un~hanged.~ An apparently more straightforward approach was the development of specific thromboxane synthase inhibitors (TSI). These drugs, in addition to inhibiting thromboxane formation, reorient the cyclic endoperoxide metabolism toward other prostaglandins, such as PGD,, and to PGI, when endothelium or leukocytes are TSI, however, do

SummaryThe combination of thromboxane synthase inhibition with thromboxane receptor antagonism has been shown to result in a strong inhibition of platelet aggregation and a prolongation of the bleeding time (Gresele et al., J. Clin Invest 1987;80: 1435–45).Ridogrel is a single molecule that efficiently achieves both inhibitions in human volunteers. The present study was performed in patients with obstructive peripheral arterial disease and elevated plasma β-thromboglobulin levels. Patients were treated with either 2 × 300 mg ridogrel or 2 × 300 mg placebo per day for 2½ days, according to a double blind randomised parallel design. Plasma β-thromboglobulin decreased significantly throughout active treatment starting within 2 h after administration; serum and urinary immunoreactive TxB2 levels and urinary 11-dehydro- TxB2 excretion were significantly lower and serum PGE2 and 6-keto-PcF1α levels significantly higher with ridogrel; no changes were observed in the placebo-treated group.In conclusion this study demonstrates a reduction of platelet activation in vivo by ridogrel.

SUMMARYProtein C deficiency has been reported to be associated with a high risk for thromboembolism. We report three patients with protein C deficiency: one suffered from left renal vein thrombosis, another from recurrent venous thrombosis of the legs complicated by pulmonary embolism and the third from an extensive thrombosis of the splanchnic veins with partial Budd-Chiari syndrome. We discuss the unusual sites of venous thrombosis in protein C deficient patients, the factors which may act as thrombotic trigger and the strategy of long-term prevention.Acta Clin Belg. 45, 2: 78–84.

SummaryClotting assays allow qualitative rather than quantitative detection of the lupus anticoagulant. We have therefore studied the usefulness of an ELISA using a commercial partial thromboplastin, Thrombofax, oS antigen; the results obtained on 146 selected patient plasmas were compared to the results of coagulation tests (kaolin clotting time, tissue thromboplastin inhibition test, activated partial thromboplastin time) and of ELISAs using cardiolipin or phosphatidylserine as antigen. While satisfactory agreement was found within the group of coagulation tests or that of ELISAs, only a moderate agreement was obtained between clotting tests and ELISAs, the best being with the partial thromboplastin ELISA using low plasma dilutions. The study further indicates that ELISA techniques cannot entirely replace coagulation tests for the detection of a lupus anticoagulant, even when a partial thromboplastin is used as antigen. On the other hand, coagulation tests are less sensitive than ELISAs for the detection of antiphospholipid antibodies.

Human muscle progenitor cells transduced with lentiviral vectors secreted high levels of blood clotting factor IX (FIX). When bioengineered into postmitotic myofibers as human bioartificial muscles (HBAMs) and subcutaneously implanted into immunodeficient mice, they secreted FIX into the circulation for >3 months. The HBAM-derived FIX was biologically active, consistent with the cells' ability to conduct the necessary posttranslational modifications. These bioengineered muscle implants are retrievable, an inherent safety feature that distinguishes this "reversible" gene therapy approach from most other gene therapy strategies. When myofibers were bioengineered from human myoblasts expressing FIX and vascular endothelial growth factor, circulating FIX levels were increased and maintained long term within the therapeutic range, consistent with the generation of a vascular network around the HBAM. The present study implicates an important role for angiogenesis in the efficient delivery of therapeutic proteins using tissue engineered stem cell-based gene therapies. ispartof: Molecular Therapy vol:14 issue:3 pages:442-451 ispartof: location:United States status: published

Aims In Belgium, general practitioners (GPs) mainly manage oral anticoagulation therapy. To improve the quality of oral anticoagulation management by GPs and to compare different models and interventions, a randomized clinical trial was performed. Methods and results Stratified randomization divided 66 GP-practices into four groups. A 6-month retrospective analysis assessed the baseline quality. In the prospective study, each group received education on oral anticoagulation, anticoagulation files, and patient information booklets (groups A, B, C, and D). Group B additionally received feedback every 2 months on their anticoagulation performance; group C determined the international normalized ratio (INR) with a CoaguChek device in the doctor's office or at the patient's home; and group D received Dawn AC computer assisted advice for adapting oral anticoagulation. For the different groups, the time spent in target INR range (Rosendaal's method) and adverse events related to anticoagulation were determined and compared with the same quality indicators at baseline. There was a significant increase in per cent of time within 0.5 INR from target, from 49.5% at baseline to 60% after implementing the different interventions. However, neither the per cent in target range nor the event rates differed among the four groups. Conclusion The interventions significantly improved the quality of management of oral anticoagulation by Belgian GPs, mainly as a result of an education and support programme.

We report here results from a United Kingdom National Quality Assessment Scheme (UK NEQAS) exercise in which both plasma spiked with monoclonal antibodies and plasma from a patient known to have lupus anticoagulant (LA) were distributed to 245 hemostasis laboratories with a request for them to test for possible LA using their routine screening procedure. In general, good agreement was seen in the diagnosis of samples spiked with monoclonal antibodies against beta2-glycoprotein 1 (beta2GP1) and prothrombin, the LA-positive patient sample, and a normal pooled plasma; over 87% of centers correctly identified each sample. However, methods employing platelet neutralizing procedures were associated with a higher proportion of false-negative responses with the antiprothrombin-spiked sample, and it is important to recognize that sensitivity and responsiveness of different methods may vary between artificial plasmas and different LA-positive patient plasmas.

Summary. This review briefly describes the development of the concepts of antiphospholipid antibody and of antiphospholipid syndrome. It focuses on the two main antigenic targets, β2 glycoprotein I and prothrombin. An excessive production of natural antibodies rather than an immune response to exogenous antigen is proposed as pathogenetic for the development of these antibodies. The review attempts to explain how some of these antibodies are anticoagulant in vitro yet prothrombotic in vivo. The final section discusses when to test for such antibodies, how to test and how to consider treatment of patients with the antiphospholipid syndrome.

Lupus anticoagulants (LAC) are a heterogeneous group of autoantibodies that prolong phospholipid-dependent clotting assays. The autoantibodies that cause LAC activity are predominantly directed against beta(2)-glycoprotein I (beta(2)GPI) or prothrombin. In the present study, we describe a method to differentiate between LAC caused by antibodies directed against beta(2)GPI or prothrombin. Monoclonal antibodies, affinity purified patient antibodies, and selected patient samples were used to show that in an aPTT-based clotting assay (PTT-LA; Diagnostica Stago), the use of cardiolipin vesicles in the neutralization procedure discriminates between beta(2)GPI- or prothrombin-dependent LAC activities. Addition of cardiolipin vesicles shortened the prolonged clotting time caused by anti-beta(2)GPI antibodies with LAC activity, whereas this procedure further prolonged clotting times caused by antiprothrombin antibodies with LAC activity. In contrast, addition of phosphatidylcholine/phosphatidylserine vesicles corrected prolonged clotting times caused by either anti-beta(2)GPI or anti prothrombin antibodies with LAC activity. The effects of cardiolipin (CL) on beta(2)GPI-induced LAC activity were specific for contact activation mediated clotting assays. Possible explanations for these findings are the relatively high affinity Of beta(2)GPI for cardiolipin, as determined by surface plasmon resonance analysis, and inhibition by anti-beta(2)GPI antibodies of the CL-induced prolongation of the PTT-LA.

Patients with prolonged clotting times caused by lupus anticoagulant (LAC) are at risk for thrombosis. This paradoxal association is not understood. LAC is frequently caused by anti-beta2-glycoprotein I (beta 2GPI) antibodies. Antibody-induced dimerization of beta 2GPI increases the affinity of beta 2GPI for phospholipids, explaining the observed prolonged clotting times. We constructed dimers of beta 2GPI that mimic effects of beta 2GPI-anti-beta 2GPI antibody complexes, and we studied their effects on platelet adhesion and thrombus formation in a flow system. Dimeric beta 2GPI increased platelet adhesion to collagen by 150% and increased the number of large aggregates. We also observed increased platelet adhesion to collagen when whole blood was spiked with patient-derived polyclonal anti-beta 2GPI or some, but not all, monoclonal anti-beta 2GPI antibodies with LAC activity. These effects could be abrogated by inhibition of thromboxane synthesis. A LAC-positive monoclonal anti-beta 2GPI antibody, which did not affect platelet adhesion, prevented the induced increase in platelet adhesion by beta 2GPI dimers. Furthermore, increased platelet adhesion disappeared after preincubation with receptor-associated protein, a universal inhibitor of interaction of ligands with members of the low density lipoprotein receptor family. Using co-immunoprecipitation, it was shown that dimeric beta 2GPI can interact with apolipoprotein E receptor 2 (apoER2'), a member of the low density lipoprotein receptor family present on platelets. These results demonstrate that dimeric beta 2GPI induces increased platelet adhesion and thrombus formation, which depends on activation via apoER2'.

Two siblings, a boy age 12 and his sister age 4 years, presented with proteinuria and hematuria, hypertension, and chronic hemolytic anemia. At age 13 years, the boy developed an episode of severe hypertensive encephalopathy and transient renal failure. Both children are attending normal school, have no neurologic symptoms, and only minimal pigmentary retinal abnormalities. Renal biopsy showed a chronic thrombotic microangiopathic nephropathy. Both patients had hyperhomocysteinemia and mild methylmalonic aciduria. Fibroblasts showed decreased cobalamin uptake, reduced methyl- and adenosyl-cobalamin formation, and deficient incorporation of formate and propionate, compatible with the Cbl-C complementation group, but milder than that found in cells from most patients. Both patients and their father carry a balanced reciprocal translocation. Parenteral hydroxycobalamin treatment reduced the homocysteine levels, and methylmalonic acid disappeared. Increasing the dosage of hydroxycobalamin from 1 to 2.5, then 5 mg daily together with betaine, further reduced homocysteine levels (boy from 118 to 23 microM and girl from 59 to 14 microM). With this treatment, hemolysis has stopped, hematuria has disappeared, proteinuria has almost normalized, and creatinine clearance has been stable. Investigations for chronic thrombotic microangiopathy should include testing for this unusual but treatable disorder, regardless of age of presentation.