Attempts to make sense of the antiphospholipid syndrome.

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.

von Willebrand factor A1 domain can adequately substitute for A3 domain in recruitment of flowing platelets to collagen.

BACKGROUND: Binding of von Willebrand factor (VWF) to platelet GPIbalpha and to collagen is attributed to VWF A1 and A3 domains, respectively. OBJECTIVES: Using VWF, VWF lacking A1 (DeltaA1-VWF) or A3 (DeltaA3-VWF) and VWF with defective A3 (H1786A-VWF), in combination with recombinant A1 (residues 1262-1492) or A3 (residues 1671-1878), fused to glutathione-S-transferase (GST-A1 and GST-A3), we have re-investigated the role of A1 in platelet recruitment to surfaces of collagen. METHODS AND RESULTS: In flow, measurable binding of DeltaA3-VWF occurred to horse tendon, but also to human type III collagen. GST-A1 and GST-A3 both competed for binding of DeltaA1-VWF and DeltaA3-VWF to horse tendon collagen fibrils in static conditions and to human collagen III during plasmon surface resonance studies, substantiating overlapping binding sites on both collagens for A1 and A3. Heparin did not affect A3-mediated binding of VWF and DeltaA1-VWF, but inhibited binding to horse tendon collagen of GST-A1 and DeltaA3-VWF. Furthermore, A1-mediated binding to type III collagen of DeltaA3-VWF binding was strongly salt-sensitive. During perfusions at wall shear rate 2500 s(-1) of calcein-labeled platelets in reconstituted blood, DeltaA3-VWF and H1786A-VWF triggered platelet binding to horse tendon collagen comparably and as potently as VWF, and to human type III collagen, only fivefold less potently, DeltaA1-VWF being inactive. Additional flow-controlled interaction studies with DeltaA3-VWF, H1786A-VWF, the collagen-VWF antagonist saratin, heparin and the VWF neutralizing antibody 82D6A3 confirmed that H1786A-VWF binds to collagen exclusively via A1. CONCLUSION: Hence, in shear forces the VWF A1 domain can assume the role of A3 to trigger substantial platelet recruitment to human collagen fibres.

Variable region heavy chain glycosylation determines the anticoagulant activity of a factor VIII antibody.

BACKGROUND: N-glycosylation occurs in the variable region of about 10% of antibodies but the role of carbohydrate at this location is still poorly understood. OBJECTIVES: We investigated the function of N-glycosylation in the variable region of the heavy chain of a human monoclonal antibody, mAb-LE2E9, that partially inhibits factor VIII (FVIII) activity during coagulation. METHODS AND RESULTS: Enzymatic deglycosylation indicated that the oligosaccharides do not determine the affinity of the antibody but enhance its FVIII neutralizing activity. A mutant antibody lacking the N-glycosylation site in the variable region of the heavy chain inhibited FVIII activity by up to 40%, while inhibition by the native antibody was 80%. To evaluate the physiological effect of such a FVIII inhibition, we investigated the ability of the mutant antibody devoid of N-glycosylation in the variable region to prevent thrombosis in mice with a strong prothombotic phenotype resulting from a type II deficiency mutation in the heparin binding site of antithrombin. Despite its moderate inhibition of FVIII activity, the mutant antibody significantly prevented thrombosis in treated animals. We also carried out glycan analysis of native and mutant antibodies. CONCLUSIONS: Modification of glycosylation in the variable region of antibodies contributes to the diversity of FVIII type II inhibition possibly by steric hindrance of the active site of FVIII by glycans, and may provide a novel strategy to modulate the functional activity of therapeutic antibodies.

The platelet ATP and ADP receptors.

Adenine nucleotides, ADP and ATP, are coreleased from dense granules during platelet activation, as well as from endothelial cells and damaged red blood cells following vascular injury. Through autocrine and paracrine mechanisms, these extracellular signaling molecules interact with the platelet P2 receptors to amplify ongoing platelet activation. Two receptors for ADP, the G(q)-protein-coupled P2Y1 and G(i)-protein-coupled P2Y12 and one receptor for ATP, the P2X1 ion channel, have been identified on platelets. Due to distinct pharmacological properties and differential regulation, the P2Y and P2X receptors essentially operate on different scales of time and distance and trigger selective intracellular signaling cascades. Recent advances in the understanding of the P2Y receptor physiology have reinforced the concept of these receptors as useful targets for antithrombotic therapy. The function of P2X1 in platelet activation only recently started to be unraveled. This review focuses on recent findings on the physiology of these platelet ADP and ATP receptors, their distinct downstream intracellular signaling pathways as well as on the available agonists, antagonists and inhibitors that allow their pharmacological discrimination.

Ambient air pollution and acute myocardial infarction.

This review summarizes the nature of ambient air pollutants, which are either gaseous or particulate of various sizes, the latter determining their penetration into the body, the smallest even translocating from the lung into the systemic circulation. It presents the epidemiological evidence linking air pollution to overall mortality, cardiovascular mortality and myocardial infarction, making the distinction between acute and chronic exposure to the pollutants. It reviews mechanistic investigations that have evaluated the links among exposure to pollutants, thrombosis, pulmonary inflammation, arterial vasoconstriction and heart rate variability. It concludes by attempting to integrate current epidemiological and mechanistic observations into a pathophysiological framework that links ambient air pollution to acute myocardial infarction and cardiovascular mortality.

The current state of clinical ethics and healthcare ethics committees in Belgium.

Ethics committees are the most important practical instrument of clinical ethics in Belgium and fulfil three tasks: the ethical review of experimental protocols, advising on the ethical aspects of healthcare practice, and ethics consultation. In this article the authors examine the current situation of ethics committees in Belgium from the perspective of clinical ethics. Firstly, the most important steps which thus far have been taken in Belgium are examined. Secondly, recent opinion by the Belgian Advisory Committee on Bioethics with regard to ethics committees is presented and the activities of Belgian ethics committees are discussed. Finally, the option to bring research ethics and clinical ethics under the roof of just one committee is criticised using a pragmatic and a methodological argument. Concomitantly, the authors build an argument in favour of the further development of ethics consultation.

[New therapeutic perspectives in hemophilia]. / Nouvelles perspectives thérapeutiques dans les hémophilies.

This paper describes the evolution of hemophilia care during the past fourty years, evolution that markedly improved the prognosis of this disorder in the developed world. Remaining problems and prospects for the future are presented.

A monoclonal antibody directed against human von Willebrand factor induces type 2B-like alterations.

We have previously described a monoclonal antibody (mAb), 1C1E7, against von Willebrand factor (VWF), that increases ristocetin-induced platelet aggregation (RIPA) and induces a preferential binding of the high-molecular-weight multimers of VWF to platelet GPIb. Further investigations using a rotational viscometer at a shear rate of 4000 s(-1) could now demonstrate that shear-induced platelet aggregation (SIPA) is significantly increased with 1C1E7 and that this could be completely inhibited by the anti-GPIb mAb 6D1. In contrast, platelet adhesion to a collagen surface at a shear rate of 2600 s(-1), using a rectangular perfusion chamber, was significantly inhibited in the presence of 1C1E7. When citrated whole blood was incubated with 1C1E7, a spontaneous binding of VWF to the platelet GPIb could be demonstrated by flow cytometric analysis. Parallel to this, a decrease of the highest molecular weight multimers of VWF in the plasma was found. Platelets with bound VWF on their surface were able to form macroaggregates but were no longer able to adhere. These phenomena are very similar to the alterations described in von Willebrand's disease type 2B. The epitope of this mAb could be localized to the N-terminal part of the subunit; therefore a distant conformational change in the A1 domain of VWF is suggested.

alphaIIbbeta3 antagonism vs. antiadhesive treatment to prevent platelet interactions with vascular subendothelium.

Platelets adhering to blood vessels promote coagulation and inflammation, and release growth factors that trigger smooth muscle cell activation. We have therefore studied the pharmacological modification of platelet deposition quantitatively by comparing adhesion of flowing platelets to various subendothelial ligands in the absence or presence of an antialpha(IIb)beta(3) antagonist with the effects of antiadhesive treatment consisting of von Willebrand factor (VWF) and fibronectin neutralization or of the combined inhibition of platelet adhesion and aggregation. In vitro, perfusion of anticoagulated human blood over calf skin collagen reiterated that alpha(IIb)beta(3) antagonism prevents platelet aggregation, but not adhesion per se: single platelets strongly bound to collagen at wall shear rates of both 1300 and 2700 s(-1), largely VWF-independent. When perfused over a human umbilical vein endothelial cell-derived extracellular matrix, single alpha(IIb)beta(3)-antagonized platelets primarily adhered to matrix-bound VWF when perfused at 2700 s(-1), but at 1300 s(-1) they also adhered significantly to fibronectin. During perfusion of anticoagulated rabbit blood over de-endothelialized rabbit aorta at a wall shear rate of 1100 s(-1), alpha(IIb)beta(3) antagonism even increased the absolute numbers of adhering platelets and VWF neutralization redirected alpha(IIb)beta(3)-antagonized platelets towards other vascular ligands. Finally, in vivo, following photochemically induced blood vessel injury in mice, alpha(IIb)beta(3) antagonism inhibited platelet-rich thrombus formation, but platelet adhesion was only significantly inhibited when associated with fibronectin neutralization. In conclusion, antiadhesive platelet treatment more potently interferes with platelet deposition on injured blood vessels than alpha(IIb)beta(3) antagonism, but abrogating platelet adhesion can only be achieved by carefully selected antiplatelet drug combinations.

Current status and implications of autoimmune antiphospholipid antibodies in relation to thrombotic disease.

This review briefly describes the development of the concepts of antiphospholipid antibody and of antiphospholipid syndrome. It focuses on the two main antigenic targets, beta2 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.

A vitamin K antagonist rapidly reverses a blue toe syndrome in a patient with lupus anticoagulant and antiprothrombin antibodies.

A 30-year old male was admitted to the hospital with extremely painful blueish discoloration of his toes. After clinical and laboratory evaluation the diagnosis of a blue toe syndrome due to primary antiphospholipid syndrome (APS) was made. Complete resolution of the blue toe syndrome occurred within 72 hours following 9 mg phenprocoumon. APS consists of the association of lupus anticoagulant or antiphospholipid antibodies with arterial or venous thrombosis, thrombocytopenia, and spontaneous abortion. The exact pathways leading to thrombosis are still unknown. Our group has previously proposed that membrane-associated immune complexes contribute towards clinical symptoms in the antiphospholipid syndrome. The case presented strengthens that concept.

Lumbar artery pseudoaneurysm and arteriovenous fistula as a complication of laparoscopic splenectomy: treatment by transcatheter embolization.

Iatrogenic injury of a lumbar artery is very rare and mostly causes retroperitoneal hemorrhage. We report a case of a lumbar artery pseudoaneurysm and a concomitant arteriovenous fistula complicating laparoscopic splenectomy and provoking renal colic-like flank pain due to mass effect on the left ureter. Definitive treatment of both vascular lesions was obtained after percutaneous transcatheter embolization of several lumbar arteries. Control computed tomography scan 3 months after embolization showed almost complete resorption of the retroperitoneal hematoma.

Air pollution and thrombosis: an experimental approach.

Air pollution is associated with cardiovascular mortality. Inhaled ultrafine particles translocate into the blood. Amine-polystyrene ultrafine particles significantly enhance experimental thrombus formation in a damaged hamster vessel and shorten the closure time in the Platelet Function Analyser. Diesel exhaust particles are thrombogenic within one hour of intratracheal instillation and shorten the closure time ex vivo. These experimental observations provide a plausible biological explanation for the epidemiologically established link between air pollution and acute myocardial infarction.

Congenital disorders of glycosylation type Ia and IIa are associated with different primary haemostatic complications.

Congenital disorders of glycosylation (CDG) type I are mostly due to a deficient phosphomannomutase activity, called CDG Ia. CDG IIa (mutations in the MGAT2 gene) results from a deficient activity of the Golgi enzyme N-acetylglucosaminyltransferase II. CDG Ia patients predominantly have a thrombotic tendency, whereas our CDG IIa patient has an increased bleeding tendency, despite similar coagulation factor abnormalities in both types. We have investigated whether abnormally glycosylated platelet membrane glycoproteins are involved in the haemostatic complications of both CDG groups. In flow cytometry, the binding of Ricinus communis lectin (reactive with beta-galactose primarily) to control platelets increased after neuraminidase treatment: this increase was smaller (p < 0.01) in CDG Ia patients (3.1 +/- 0.08 times) than in control platelets (8.5 +/- 1.8 times) and did not occur in the CDG IIa patient. Platelet-rich plasma from CDG Ia patients, but not a CDG IIa patient. aggregated spontaneously and gel-filtered platelets from CDG Ia patients agglutinated at very low concentrations of ristocetin, independently of von Willebrand factor (vWF). Accordingly, in stirred whole blood, the rate of single platelet disappearance of CDG Ia patients was twice that of control platelets. In contrast, perfusion of whole anticoagulated blood of the CDG IIa patient over collagen yielded markedly decreased platelet adherence to collagen at shear rates involving glycoprotein (GP) Ib-vWF interactions. Thus, abnormal glycosylation of platelet glycoproteins in CDG Ia enhances nonspecific platelet interactions, in agreement with a thrombotic tendency. The reduced GP Ib-mediated platelet reactivity with vessel wall components in the CDG IIa patient under flow conditions provides a basis for his bleeding tendency.

Genetic variation of the extra-large stimulatory G protein alpha-subunit leads to Gs hyperfunction in platelets and is a risk factor for bleeding.

Alternatively spliced GNAS1 and XL-GNAS1, encoding respectively the stimulatory G-protein alpha-subunit (Gsalpha) and the extra-large stimulatory G-protein alpha-subunit (XLsalpha), 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 XLsalpha 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 XLsalpha insertion represents a new genetic cause of an inherited bleeding tendency, although to a variable degree.