Follow-up of babies born to mothers with antiphospholipid syndrome: preliminary data from the European neonatal registry.

In this review preliminary data on the follow-up of 141 babies born to mothers with antiphospholipid syndrome are reported. In spite of maternal treatment, the rate of both preterm delivery and low birth weight were 16 and 17%, respectively. At birth, no clinical evidence of perinatal thrombosis was observed. Placental transfer of antiphospholipid antibodies occurred in 20, 25 and 43% of cases for lupus anticoagulant, anticardiolipin and anti-ß2-glycoprotein I antibodies, respectively. At 24 months of follow-up, four children showed behaviour abnormalities suggesting the possible need for long-term neurological evaluation in this clinical setting.

Morbidity and mortality in the antiphospholipid syndrome during a 5-year period: a multicentre prospective study of 1000 patients.

OBJECTIVES: To identify the main causes of morbidity and mortality in patients with antiphospholipid syndrome (APS) during a 5-year period and to determine clinical and immunological parameters with prognostic significance. METHODS: The clinical and immunological features of a cohort of 1000 patients with APS from 13 European countries who had been followed up from 1999 to 2004 were analysed. RESULTS: 200 (20%) patients developed APS-related manifestations during the 5-year study period. Recurrent thrombotic events appeared in 166 (16.6%) patients and the most common were strokes (2.4% of the total cohort), transient ischaemic attacks (2.3%), deep vein thromboses (2.1%) and pulmonary embolism (2.1%). When the thrombotic events occurred, 90 patients were receiving oral anticoagulants and 49 were using aspirin. 31/420 (7.4%) patients receiving oral anticoagulants presented with haemorrhage. 3/121 (2.5%) women with only obstetric APS manifestations at the start of the study developed a new thrombotic event. A total of 77 women (9.4% of the female patients) had one or more pregnancies and 63 (81.8% of pregnant patients) had one or more live births. The most common fetal complications were early pregnancy loss (17.1% of pregnancies) and premature birth (35% of live births). 53 (5.3% of the total cohort) patients died. The most common causes of death were bacterial infection (21% of deaths), myocardial infarction (19%) and stroke (13%). No clinical or immunological predictor of thrombotic events, pregnancy morbidity or mortality was detected. CONCLUSION: Patients with APS still develop significant morbidity and mortality despite current treatment (oral anticoagulants or antiaggregants, or both).

A phospholipase A2 with anticoagulant activity. II. Inhibition of the phospholiped activity in coagulation.

An anticoagulant factor with phospholipase A2 activity has been isolated from Vipera berus venom. Phospholipase activity was studied on platelet phospholipid and on brain cephalin. The venom factor showed a potent anticoagulant activity: 1 mug impaired the clotting of 1 ml of citrated recalcified platelet-poor plasma. The anticoagulant inhibited clotting by antagonism to phospholipid. The antagonism constant (Kan = 6.8-10(-9) M) demonstrated the high affinity of the inhibitor for phospholipid. As with other phospholipases A2, the venom factor was thermoresistant but very sensitive to photo-oxidation. Both activities (anticoagulant activity and phospholipase activity) were not markedly dissociated by either denaturation or neutralization processes. Slightly different curves of photo-oxidative inactivation of both activities suggested the presence, on the molecule, of two very close sites responsible for phospholipase and anticoagulant activities. The inhibitor effect on coagulation was independent of the hydrolysis process. In fact, lysoderivatives and fatty acids, resulting from complete hydrolysis with the venom factor, were as active as the native phospholipids. Moreover phospholipase A2 from other viperidae venom, which did not have anticoagulant activity, produced similarly active lysoderivatives. This showed that the cleavage of the beta-acyl bond does not interfere with the activity of phospholipid. A possible mechanism of clotting inhibition by the venom factor was proposed. Owing to its high affinity for phospholipid, the inhibitor would complex phospholipid at its protein binding site impairing the normal arrangement of coagulation protein factors and, consequently, their activation. The positive charges of the inhibitor (pI = 9.2) could bind with phosphoryl or carboxyl groups of phospholipid, making them unavailable for protein binding. The complex formation involves a loss of dissociating capacity of the enzyme towards its substrate. This required an additional interaction of the inhibitor with a coagulation protein factor. The inhibitor could be removed from the complex by specific antibodies, permitting recovery of normal phospholipid-protein interaction. The role of calcium in the complex has not yet been elucidated. This venom factor affords a useful tool for investigating the phospholipid-clotting protein interaction.

A phospholipase A2 with anticoagulant activity. I. Isolation from Vipera berus venom and properties.

An anticoagulant protein has been isolated by DEAE cellulose chromatography and gel filtration from the venom of the Vipera berus orientale (Eastern Europe). Purification has been completed by elution on carboxymethyl cellulose with continuous gradient at constant pH. The inhibitor of coagulation was separated from the other venom enzymes, e.g. procoagulant, fibrinogenolytic, aminoesterase and amino acid oxidase activities. It was also separated from other phospholipase components which were not related to the anticoagulant property. The inhibitor appeared as a simgle polypeptidic chain protein, formed by 119 amino acid residues, with a molecular weight of 13400 and an isoelectric point of 9.2. At low saline molarity, a monomer-trimer transition of this protein was observed. Both forms had the same amino acid composition. There were six disulfide bridges without free SH groups per phospholipase molecule. Deprived of any proteolytic activity, the clotting inhibitor displayed a high phospholipase activity in the presence of calcium. Activity did no appear with EDTA buffer deprived of cation. Finely dispersed micellar suspensions were found suitable for obtaining the highest phospholipase activity. High sodium cholate concentration or methanol/chloroform/ether solvent were effective without loss of enzymatic activity. As characteristis of phospholipase A2 (EC 3.1.1.4), the degradation products identified on thin-layer chromatography induced hemolysis of human erythrocytes. The apparent Km value 1.25 - 10(-3) M was determined on phosphatidylcholine isolated from ovolecithin. This purified berus inhibitor would be of value for investigating the involvement of phospholipids in the clotting mechanism.

Plasma levels of thrombomodulin in pulmonary hypertension.

BACKGROUND: Long-standing pulmonary hypertension (PH) leads to structural alterations of the pulmonary vasculature and its endothelium, and occlusion of small vessels by microthrombi. In patients with PH, the search for factors inducing or worsening endothelium damage and in situ thrombi is still ongoing. Thrombomodulin (TM), an endothelial cell membrane protein, is a receptor for thrombin and a major anticoagulant proteoglycan. PURPOSE: To analyze plasma TM levels in patients with different forms of severe PH. PATIENTS: We prospectively studied 32 consecutive patients with PH referred for heart, lung, or heart-lung transplantation: 11 patients with primary PH (group 1), 11 patients with secondary precapillary PH (Eisenmenger's syndrome, group 2) and 10 patients with secondary postcapillary PH due to congestive heart failure (group 3). Thirty-eight healthy subjects were also studied as a control group. METHODS: Plasma concentrations of TM were measured by an immunoenzymatic technique that uses two anti-TM monoclonal antibodies that have a strong avidity and react with different epitopes of the molecule. RESULTS: Thrombomodulin plasma levels decreased in all patients with precapillary PH, and this decrease was highly significant compared with controls (26 +/- 2 versus 44 +/- 2 ng/mL, P = 0.0001). In primary PH, the TM decrease was only significant in males whereas in the Eisenmenger's syndrome TM values were the lowest of all the patients studied, with mean values twice as low as controls (22 +/- 2 versus 44 +/- 2 ng/mL, P = 0.0001). In contrast, in postcapillary PH, studied only in males, TM levels were increased (85 +/- 17 versus 54 +/- 3 ng/mL, P = 0.02). Patients with precapillary PH had more severe disease than patients with postcapillary PH, with higher pulmonary artery pressure and pulmonary vascular resistance (P < 0.001). There was no correlation between TM plasma levels and all hemodynamic variables. CONCLUSION: We found low levels of plasma TM in patients with precapillary PH but not in postcapillary PH compared with healthy controls. This may be related to the severity of PH and may contribute to the initiation or worsening of in situ thromboses frequently found in pulmonary hypertension. Further studies should analyze whether other markers of endothelial cell damage are correlated with plasma TM levels in patients with precapillary pulmonary hypertension.

Plasma thrombomodulin in orthotopic liver transplantation.

Plasma thrombomodulin (TM), a specific marker of vascular endothelial injury was measured pre-, per-, and postoperatively in 16 consecutive patients undergoing orthotopic liver transplantation (OLT). The TM level, which was already elevated preoperatively, remained unchanged during OLT, except for an acute and transitory spike at the time of graft reperfusion. This TM peak is probably attributable to an acute release from the patient's endothelium because the TM level in the last saline rinse of the graft before implantation was low. This TM spike was not correlated with the progressive tissue-type plasminogen activator (t-PA) increase, plasminogen activator inhibitor 1 (PAI-1), or von Willebrand factor (vWF) values. The absence of accumulation of TM in plasma, unlike that of t-PA, suggests that the liver does not play a major role in TM clearance in humans. At the end of surgery, individual TM values returned to preoperative levels and remained unchanged during the 7 days following surgery. This observation suggests that the high (or very high) TM levels measured in these patients might be due to an indirect rather than a direct effect of liver dysfunction on the vascular endothelium which remained damaged during the postoperative period. The possibility that TM might be a predictive marker for thrombotic OLT complications remains to be investigated in a postoperative follow-up study.

Preservation of thrombomodulin antigen on vascular and extravascular surfaces.

The protein C anticoagulant system is mediated by thrombin and is highly accelerated by thrombomodulin. We studied the distribution of thrombomodulin antigen (TM Ag) in the rabbit using an affinity-purified antibody raised in a goat against rabbit thrombomodulin. The preservation of TM Ag was highly dependent on immediate fixation of the surface on which it is located. TM Ag was found on the endothelium of the entire vasculature, whereas it was absent from all connective tissue, smooth and striated muscle, secretory epithelia, cartilage, bone, neural tissue, and all parenchyma examined. A new finding was the presence of TM Ag on nonvascular surfaces of body cavities (the mesothelia of pleura, pericardium, and peritoneum, the synovial membrane, and the arachnoid enveloping the central nervous system). By use of a functional assay, TM activity was recovered in buffered saline/detergent solution which was either injected into the intraperitoneal cavity of rabbits in vivo or incubated with the surface of the arachnoid in vitro. These findings extend the importance of anticoagulant mechanisms to the systems of slowly circulating fluids, in which they might be required for maintenance of the flow, and to mesothelial cavities, in which they could be necessary for preventing adherence between the surfaces, in conditions associated with pathological exudation.

Increased plasma thrombomodulin in cancer patients.

Plasma samples from 35 patients with colorectal cancer, 16 patients with pancreatic cancer and 46 patients with various cancers in the terminal stage were analysed for soluble plasma thrombomodulin with an ELISA method. At time of diagnosis and before primary treatment, the patients with colorectal cancer had normal plasma TM levels. In the patients who developed disseminated disease, the mean plasma TM level increased significantly. In the patients with pancreatic cancer, the mean plasma TM level was increased already at time of primary treatment. The TM level increased further with progress of the pancreatic cancer. In the patients with various cancer types in the terminal stage, the mean TM was also significantly increased compared to healthy controls. Great individual variation in the plasma TM level was observed, as well as great variation of mean TM level between the various cancer types. There was no significant correlation between the TM levels and the levels of tissue factor pathway inhibitor, another endothelial coagulation inhibitor, which increased with progress of malignant disease. This may indicate different underlying mechanisms for the increased plasma levels.

Plasma contact activation and decrease of factor V activity on negatively-charged polyelectrolytes.

The effect of negatively-charged polymers, used in some artificial devices, on plasma clotting and kinin systems was studied in vitro using polyelectrolyte complexes. Contact activation was observed as an immediate, transient and surface-dependent phenomenon. After incubation of the plasma with the polymer a small decrease of factor XII activity was noticed, which corresponded to a greater reduction of prekallikrein activity and to a marked kinin release. No significant decrease of factor XII, prekallikrein, HMW kininogen could be detected immunologically. Only the initial contact of the plasma with the polyelectrolyte lead to activation, subsequently the surface became inert. Beside contact activation, factor V activity also decreased in the plasma. The decrease was surface and time-dependent. It was independent of contact factor activation, and appeared to be related to the sulfonated groups of the polymer. If purified factor V was used instead of plasma factor V, inactivation was immediate and not time-dependent suggesting a direct adsorption on the surface. A second incubation of the plasma-contacted polymer with fresh plasma resulted in a further loss of Factor V activity.

Purification and properties of prothrombin modified by asbestos filtration of human plasma.

Filtration through asbestos filter (Seitz) of human plasma modified the prothrombin molecule as previously shown. Factor II could no longer be activated by physiological activators (Ca++ + phospholipid + V and Xa) but reacted readily with staphylocoagulase. The separation and purification of the modified prothrombin allowed allowed the preparation of two fractions: A small slightly modified accessory fraction, "prothrombin-asbestos-1", lost its ability to be activated by physiological activators, and its ability to be adsorbed by barium citrate, but retained the immunological reactivity of fragment 1, as well as the mobility and molecular weight of unmodified prothrombin. A main fraction, "prothrombin-asbestos-2" appeared to be a modified prothrombin which has lost its N-terminal extremity. It was not adsorbed by barium citrate and could not be activated by physiological activators. It possessed a reduced electrophoretic mobility, as well as a reduced molecular weight (39,000), which are properties similar to those of thrombin. Both fractions 1 and 2 were devoid of thrombin activity. Asbestos was thus able to break the prothrombin molecule non enzymatically, the amputation of the N terminal extremity being responsible for the new functional and physicochemical properties of the molecule. Staphylocoagulase appeared not to need the N terminal extremity of the molecule of prothrombin to form the active thrombin-coagulase complex.

Interactions between human plasma proteins and heparin-poly(methyl methacrylate) copolymer.

A solid Heparin-PMMA copolymer has been synthetized by a radical polymerization of methyl methacrylate from oxidative reaction initiated by Ce4+ ions in the presence of heparin. Covalently linked heparin was 10% of copolymer weight. The antithrombin activity of the copolymer corresponded to 1% of grafted heparin. PMMA sequence of the copolymer played the leading role in fibrinogen, immunoglobulins, transferrin and albumin adsorption. These proteins adsorbed on the copolymer, showed different competitive desorption pattern in the presence of whole plasma: fibrinogen presented the highest degree of affinity for the copolymer. The heparin part of the copolymer was responsible for antithrombin III adsorption and for decrease of factor V activity. Active antithrombin III was eluted. An inactivation of factor V in plasma was observed using high concentrations of soluble heparin. This result suggested that copolymer heparin chains, even devoid of antithrombin activity were involved in this inactivation. With Heparin-PMMA copolymer, plasma clotting pro-enzymes behaved differently than on heparin-sepharose copolymer:disappearance of factor XI activity, decrease in prekallikrein activity and activation of factor IX were observed. PMMA sequences were responsible for factor IX activation.

Platelet hyperactivation in patients with essential thrombocythemia is not associated with vascular endothelial cell damage as judged by the level of plasma thrombomodulin, protein S, PAI-1, t-PA and vWF.

The occurrence of thrombotic events remains an important clinical problem in Essential Thrombocythemias (ET). Thus, hemostatic, fibrinolytic and vascular status was investigated in 16 patients (5 males and 11 females) with ET. Among them five presented thromboses in their past history. Platelet hyperactivation, as evidenced by a mean three-fold increase in plasma betathromboglobulin (beta TG), was observed in 13 among 16 patients; surprisingly this activation was present even when the platelet count was normal (in two patients) or subnormal, below 600 x 10(9)/l (in 11 patients). The mean value was 104 +/- 57 IU/ml significantly different from that of normal controls (35 +/- 16.5 IU/ml) (p < 0.001). An artefactual in vitro platelet activation was ruled out by the concomitant measurement of platelet factor 4 (PF4). D-dimers fibrin degradation products (D-Di FDP) were normal in all patients. Vascular endothelial cell function parameters were not markedly modified. The mean value of plasma thrombomodulin (TM) was found slightly but not significantly increased (60.1 +/- 4.9 ng/ml versus 49.1 +/- 10.0 ng/ml in controls). The values of plasma TM correlated neither with that of the platelet count nor with that of plasma beta TG or plasma PF4. The mean values of plasma protein S, von Willebrand factor (vWF), plasminogen activator inhibitor type 1 (PAI-1), tissue plasminogen activator (tPA) were normal and were not correlated neither with that of plasma TM nor with that of plasma beta TG.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro thrombogenicity tests of factor IX concentrates. II: effects of phospholipids and heparin.

Measurement of the total phospholipid (and that portion active in coagulation) in factor IX concentrates revealed no correlation with in vitro tests of potential thrombogenicity, except in the case of the recalcification time and the thrombin generation test which may detect coagulant phospholipid as well as the presence of thrombogenic enzymes. This is probably due to separation of the prothrombin complex proteins from most phospholipid during ion-exchange chromatography. Although low levels of phospholipid remain in the final product these are apparently insufficient to effect appreciable activation of factor IX concentrates despite low levels of antithrombin III. Two tests which measure the formation of thrombin and factor Xa after recalcification of concentrates were affected by the addition of exogenous phospholipid. However this is a relative effect such that differences are quantitative rather than qualitative. Heparin addition during production of factor IX concentrate was found to have only minor effects on the results of in vitro thrombogenicity tests of the final product. This was confirmed in the laboratory by incubation of unheparinised products with heparin for periods of up to 6 hr.

Studies on prothrombin complex concentrates contact factors, complement components and proteinase inhibitors.

The behaviour of contact factors, complement components and antiproteases during the preparation of prothrombin complex concentrates by adsorption of the clotting components on DEAE-Sephadex has been studied. The pro-enzymes: factors XII, XI and prekallikrein were removed by pre-elution in function of the salt concentration. In contrast, high molecular weight kininogen was considerably enriched in PCC preparations. C4 of the complement system displayed an analogous behaviour. C1s reached a 4-5 fold plasma concentration but C3 only 30% of the normal plasma level. The prothrombin complex concentrate contained no antithrombin III nor alpha 2M nor alpha 2 antiplasmin but a three fold plasma concentration of C1-inactivator and a 15 fold increase of inter-alpha-trypsin inhibitor. NAPTT (Non Activated Partial Thromboplastin Time) ratios did not seem to be in accordance with either the presence or the absence of contact enzymes. Moreover 0.20 M NaCl appeared as the minimal pre-elution molarity necessary to ensure a NAPTT ratio above thrombogenic values. Molecular alteration of high molecular weight kininogen and C4 was observed and its significance discussed. Complex formation between C1-inactivator and proteases was shown to be another sign of undesirable proteolytic events.

Anticardiolipin antibody assay: a methodological analysis for a better consensus in routine determinations--a cooperative project of the European Antiphospholipid Forum.

Despite the widely recognized practical importance of anticardiolipin (aCL) ELISA, the reliability of this test has been recently discussed. In order to investigate this area on European scale, we sent to 30 experienced centers a questionnaire focusing on the diagnostic procedures applied to patients with antiphospholipid syndrome (APS) and on the detailed protocols used to perform aCL. Anticardiolipin ELISA was found to be the most frequently performed test in patients with suspected APS, but significant difference was shown among the various protocols. The cross-laboratory multiple examination of ten serum samples evaluated independently by the 24 centers pointed out the difficulty in getting comparable results. Therefore a "consensus" protocol was derived from the aCL methods giving the best performance. The materials and reagents necessary to perform the "consensus" method, including, as putative standards, one IgG and one IgM monoclonal antibody (HCAL and EY2C9) were distributed to 19 Centers. The results of one IgG and one IgM aCL high positive sera measured in serial dilutions were compared. A progressive decrease in the variability of the values obtained for a given sample appeared evident when all the laboratories used the same standard, in their own in-house ELISA and even more in the "consensus" ELISA. Our data show that aCL ELISA standardization is necessary in order to obtain comparable results in different laboratories.

Predictive value of plasma thrombomodulin in preeclampsia and gestational hypertension.

In a prospective longitudinal study, 130 primigravidae at risk for preeclampsia were examined and plasma sampling performed in 45 of them. Plasma thrombomodulin (pTM) was sequentially measured at weeks 12, 24 and 32 of gestation and after delivery in 20 primigravidae who developed either mild preeclampsia (n = 8) or gestational hypertension (n = 12) between weeks 32 and 39 of gestation and in 25 (age-matched) primigravidae who had uneventful pregnancies. pTM elevations were not observed until week 32 in uneventful pregnancies, but were present by week 24 (p = 0.002) in patients who later developed hypertensive complications. A net individual pTM increase > or = 4.2 ng/ml between weeks 12 and 24 (more than 8 times that of normotensive primigravidae) and/or pTM level > or = 47.5 ng/ml at week 32 predicted the development of hypertensive complications with 80% accuracy. Serial pTM determinations can be useful to select pregnancies who may benefit from early pharmacological intervention.

High levels of circulating thrombomodulin in human foetuses and children.

Thrombomodulin (TM) is an endothelial cell surface proteoglycan with anticoagulant functions, also implicated in cell proliferation, cell-cell adhesion and differentiation. In this study we determined circulating plasma TM (pTM) levels in human foetuses at different stages of pregnancy, at birth and in childhood. TM levels increased with gestational age, the median level reaching a peak of approximately 165 ng/ml between the 23rd and 26th week, thereafter decreasing gradually, reaching a value of 108 ng/ml at birth. pTM continues to decrease progressively during childhood, reaching in the 5-15 years group a median of 56 ng/ml which approaches the adult value. The pTM peak was statistically significant and represents a specific foetal phenomenon as it was independent of the corresponding maternal values. As a whole, the pTM pattern during foetal maturation appears totally different from that of protein C, prothrombin and other coagulation activators and inhibitors and thus, TM may play in the foetus another role in addition to its well-known anticoagulant function.

Plasmodium falciparum-infected erythrocytes: a mutational analysis of cytoadherence via murine thrombomodulin.

The pathophysiologic events leading to organ damage in Plasmodium falciparum malaria infections involve adhesion and sequestration of parasite-infected erythrocytes (PRBC) to the vascular endothelium and syncytiotrophoblast. Several potential receptors to which the PRBCs may bind have recently been identified, one of which is thrombomodulin (TM). TM has been implicated particularly in mediating sequestration of P. falciparum-infected erythrocytes in the placenta and brain, two sites of disease associated with high morbidity. In order to establish that binding of parasite-infected red blood cells to TM is dependent on its containing chondroitin-4-sulfate (CSA), we have mutated the CSA-attachment site of murine TM, and expressed this mutant form (TMsergly) in COS-7 cells. In cytoadhesion assays, we demonstrate that, in contrast to wild-type TM which contains CSA and supports the adhesion of 1466 PRBCs/mm2, TMser-gly does not contain CSA and adhesion of PRBCs to those cells expressing TMser-gly is entirely abrogated (200 PRBCs/mm2). These studies further confirm that the CSA of TM may play a role in the pathophysiology of malaria by providing a binding site for PRBCs.

Antiphospholipid antibody production during Mediterranean spotted fever.

Antiphospholipid antibodies (aPLA) were discovered during the course of Mediterranean spotted fever (MSF) caused by Rickettsia conorii and characterized by endothelial cell (EC) damage resulting from this organism's tropism for EC. In two MSF patients, two types of aPLA were identified: antiphosphatidylethanolamine antibodies detected by immunological methods and lupus anticoagulant detected by clotting assays. The persistence of both aPLA for several months after the acute phase and clinical recovery might correspond to a durable immunological response to membrane damage of EC caused by R. conorii. Their possible role in the pathophysiology of microthrombi formation observed during MSF remains to be elucidated in a study on a larger number of patients.