Extracellular protein disulfide isomerase regulates feedback activation of platelet thrombin generation via modulation of coagulation factor binding.

BACKGROUND: Protein disulfide isomerase (PDI) controls platelet integrin function, tissue-factor (TF) activation, and concentrates at fibrin and thrombus formation sites of vascular injury. OBJECTIVE: To investigate the involvement of surface thiol isomerases and especially PDI, in thrombin-mediated thrombin amplification on human platelets. METHODS/RESULTS: Using a newly developed thrombin-dependent platelet thrombin generation assay, we observed that the feedback activation of thrombin generation on the platelet surface does not depend on TF, as anti-TF antibodies inhibiting TF-induced thrombin formation in platelet-depleted plasma had no effect compared with vehicle-treated controls. Feedback activation of thrombin generation in the presence of platelets was significantly diminished by membrane impermeant thiol blockers or by the thiol isomerase-inhibitors bacitracin and anti-PDI antibody RL90, respectively. Platelet thrombin formation depends on binding of coagulation factors to the platelet surface. Therefore, involvement of thiol isomerases in this binding was investigated. As shown by confocal microscopy and flow cytometry, thrombin-stimulated platelets exhibited increased surface-associated PDI as well as extracellular disulfide reductase activity compared with unstimulated platelets. Flow cytometric analysis revealed that membrane impermeant thiol blockers or PDI inhibitors, which had been added after platelet stimulation and after phosphatidylserine exposure to exclude their influence on primary platelet activation, significantly inhibited binding of all coagulation factors to thrombin-stimulated platelets. CONCLUSIONS: Thus, surface-associated PDI is an important regulator of coagulation factor ligation to thrombin-stimulated platelets and of subsequent feedback activation of platelet thrombin generation. Cell surface thiol isomerases might be therefore powerful targets to control hemostasis and thrombosis.

Changes in autoimmune markers of the anti-cardiolipin syndrome on days of extreme geomamagnetic activity.

UNLABELLED: The possible relation between various biological or medical phenomena and changes in environmental physical activity, such as Solar, Geomagnetic Activity (GMA); Cosmic Ray; Proton, and other particle flux, have been reported. These phenomena seem to be reflected, among others, in the immune system, resulting in changes in immunoglobulin (Ig) levels or outbursts of epidemics. AIM: to examine a possible association of GMA with another aspect of the immune system--autoimmunity. Fluctuations of levels of anticardiolipin (IgG; IgM, IgA subtypes) and lupus anticoagulant (Kaulin clotting time and Dilute Russell's viper venom time) autoantibodies, serving as anticardiolipin syndrome (ACLS) markers, were monitored during days of severe GMA storms and compared with those of lowest/quiet GMA days. Cosmophysical data were obtained from the NOAA National Space Service Center and the National Geophysical Data Center, USA. RESULTS: A significant rise in the levels of anti beta2Gp1-IgA (p = 0.0001); and KCT (p = 0.019) was observed on days of the GMA storms. CONCLUSION: On days of major GMA storms, significant changes in the autoimmune marker levels of ACLS were observed compared with quiet days. An involvement of those changes in clinical events related to GMA storms is possible.

Function-modulating human monoclonal antibodies against platelet-membrane receptors isolated from a phage-display library.

Monoclonal antibodies to platelet membrane receptors have been used extensively for analysis of receptor structure and function. Function-blocking human antibodies are being used for the development of antiplatelet drugs. We isolated human monoclonal antibodies from a library of single-chain Fv (scFv) antibodies displayed on the surface of filamentous phage, by selection on whole platelets. Eight different platelet-binding clones were isolated, of which three bound to the platelet-membrane glycoprotein (GP) GPIb in an ELISA assay. Specific elution with a recombinant polypeptide of von Willebrand factor (VWF) spanning the GPIbalpha binding site, yielded the same three phage clones. Two of the three anti-GPIb clones could be purified as scFv monoclonal antibodies, and they competed with each other for binding to intact platelets, suggesting that they bind at or near the same site on GPIb. Their binding affinities differed, however, and the clone with higher affinity inhibited ristocetin-induced platelet aggregation. These data indicate that selection from a phage display library of human scFvs using whole platelets can be applied for the isolation of functional antiplatelet-GPIb antibodies useful for the development of new therapeutic and diagnostic strategies.

Effect of thrombophylaxis on uterine and fetal circulation in pregnant women with a history of pregnancy complications.

PURPOSE: The aim of this study was to investigate the effect of thromboprophylactic therapy on fetal and maternal Doppler flow parameters in pregnant women with severe complications in previous pregnancies and evidence of acquired or congenital thrombophilia in the current pregnancy. METHODS: Sixty-five patients with a history of recurrent abortions, intrauterine fetal death, intrauterine growth restriction (IUGR), and severe early-onset preeclampsia were tested for the presence of acquired or congenital thrombophilia. Those with positive findings were prescribed low-dose aspirin plus low-molecular-weight heparin (LMWH) (enoxaparin); the remainder received low-dose aspirin only. A Doppler flow study was performed before and after treatment and in the third trimester of pregnancy. RESULTS: Of the 65 pregnancies, four ended in spontaneous abortion and were excluded from the analysis. Of the 61 women with completed pregnancies, 37 (61%) had evidence of acquired or congenital thrombophilia: 22 (36%) protein S deficiency; 1 (2%) protein C deficiency; 2 (3%) activated protein C resistance (APC-R); 2 (3%) IgG for antiphospholipid antibodies; 1 (2%) circulating anticoagulant; and 9 (15%) a combined defect. This group showed a significant decrease in mean uterine artery pulsatility index (PI) before and after treatment (1.32+/-0.36 vs. 1.04+/-0.23, P=.006), whereas the remaining 24 patients treated with low-dose aspirin only had nonsignificant changes. Pearson's correlation test yielded no correlations of the pregnancy outcome parameters with Doppler flow values in the umbilical or uterine arteries. CONCLUSIONS: Thromboprophylactic therapy transiently improves maternal circulation parameters in patients with thrombophilia at risk of fetal loss and other severe complications of pregnancy, but not in correlation with their pregnancy outcome. Therefore, Doppler examination of maternofetal circulation in the second trimester is not predictive of pregnancy outcome.

Changes in the effects of interleukin-1beta and tumor necrosis factor-alpha on platelet activation in early pregnancy.

Platelets are known to be activated in normal pregnancy, and are further activated in pathological pregnancy states, such as preeclampsia. The factors controlling platelet activation are unknown, but cytokines, such as interleukin 1beta (IL-1beta) and tumor necrosis-alpha (TNF-alpha) have been found to affect platelet function and are believed to be involved in early pregnancy. We assessed the effects of these cytokines on platelets from women at various stages of pregnancy. We compared two methods: platelet in vitro aggregation by aggregometry, and platelet P-selectin expression by flow cytometry. IL-1beta and TNF-alpha had no effect on the in vitro aggregation and P-selectin expression of platelets from women in the first trimester of pregnancy as compared to the inhibitory effects of both in late pregnancy. We conclude that maternal platelet function undergoes a marked change throughout pregnancy.

Regulation of platelet aggregation and adenosine triphosphate release in vitro by 17beta-estradiol and medroxyprogesterone acetate in postmenopausal women.

Clinical studies have suggested that hormone replacement therapy (HRT) may reduce the risk of coronary heart disease in postmenopausal women. Although progestins are commonly added to HRT preparations for uteroprotection, the perceived beneficial cardiovascular effects of HRT are thought to be mediated predominantly by the estrogen component. Platelets play a critical role in the pathogenesis of atherosclerosis and cardiovascular disease and, hence, it is possible that the cardiovascular effects of estrogens are mediated, at least in part, through inhibition of illicit platelet activation. The aim of this study was to examine the effects of sex steroids on adenosine diphosphate (ADP)-induced platelet aggregation and adenosine triphosphate (ATP) release in vitro in postmenopausal women. In addition, the effects of antiestrogens 14-hydroxy tamoxifen (4-OHT) and ICI 182780] and antiprogestins (RU 486 and ZK 98299) were also investigated. Preincubation of platelet-rich plasma (PRP) with antiestrogens or antiprogestins did not alter subsequent platelet aggregation or ATP release in response to ADP. However, preincubation with 17beta-estradiol (E2) significantly inhibited ADP-mediated platelet aggregation by a mean (+/-SEM) of 37%+/-6% (p = 0.02) and ATP release by 82%+/-6% (p = 0.03), an effect that was reversed by the addition of ICI 182780 or 4-OHT but not RU 486 and ZK 98299. Although the progestin medroxyprogesterone acetate (MPA) also significantly inhibited platelet aggregation (by 28%+/-5%, p = 0.02) and ATP release (by 63%+/-9%, p = 0.02), this inhibition was not reversed by the addition of antiprogestins or antiestrogens. These data show that sex steroids can modulate platelet function in vitro. Furthermore, as platelets are devoid of nuclear components, these findings indicate that estrogens may regulate platelet function through binding to a non-nuclear receptor with ligand-binding properties similar or identical to the wild-type receptor. By contrast, MPA appears to exert its effect through a mechanism that does not involve binding to the "classical" progesterone receptor.

Increased D-dimer levels in twin gestation.

In normal pregnancy, the hemostatic balance is displaced toward hypercoagulability. The elevation in plasma levels of coagulation factors VII, VIII, and X and fibrinogen and the increased concentrations of plasminogen activator inhibitors [1,2] may predispose individuals to thromboembolism, especially near term [1,3]. Because human multifetal gestation requires still greater physiological alterations, the imbalance in hemostasis is further exaggerated. It has been suggested that the changes in the coagulation system near term may even mimic low-grade disseminated intravascular coagulopathy [4]. However, for the majority of women with multifetal gestation, the coagulopathy observed in the laboratory is not clinically apparent [5]. Despite the large body of research on the physiological adaptation to pregnancy, relatively little is known of the biological adaptation in general and the hemostatic changes in particular associated with multiple gestation.

Protein disulfide isomerase mediates integrin-dependent adhesion.

Cell adhesion is mediated by the integrin adhesion receptors. Receptor-ligand interaction involves conformational changes in the receptor, but the underlying mechanism remains unclear. Our earlier work implied a role for sulfhydryls in integrin response to ligand binding in the intact blood platelet. We now show that non-penetrating blockers of free sulfhydryls inhibit beta(1) and beta(3) integrin-mediated platelet adhesion regardless of the affinity state of the integrin. Removal of the inhibitors prior to adhesion fully restores adhesion despite the irreversible nature of inhibitor-thiol interaction, indicating sulfhydryl exposure in response to adhesion. We further show that blocking protein disulfide isomerase (PDI) inhibits adhesion. These data indicate that: (a) ecto-sulfhydryls are necessary for integrin-mediated platelet adhesion; (b) disulfide exchange takes place during this process; (c) surface PDI is involved in integrin-mediated adhesion.

Low-molecular-weight heparin for thrombophilia in pregnant women.

OBJECTIVE: Low-molecular-weight heparin (LMWH) is the anticoagulant of choice during pregnancy because it is associated with a low incidence of osteoporosis and thrombocytopenia. Antithrombotic therapy has recently been used to prevent pregnancy loss in high-risk patients with evidence of acquired or congenital thrombophilia. The aim of the present study was to gain further information on the teratogenic potential of LMWH in this patient group. METHODS: The study population included 46 patients with a history of recurrent abortions, intrauterine fetal death or intrauterine growth restriction (IUGR) and severe early-onset preeclampsia. Patients with a history of thromboembolism or positive findings for thrombophilia were prescribed LMWH (enoxaparin sodium, 40 mg daily) in combination with low-dose aspirin (100 mg daily) in the first trimester (group 1, n=14) or the second trimester (group 2, n=17); the remaining 15 patients received low-dose aspirin alone (group 3). RESULTS: No significant differences were noted between the groups in the incidence of congenital malformations or abortions, IUGR or preterm deliveries. One infant in group 1 had familial bilateral postaxial polydactyly of the hands and one in group 3 had patent ductus arteriosus. CONCLUSION: Despite the small size of the study groups, our results support the assumption that the use of LMWH is safe, at least as a teratogenic agent, in patients with thrombophilia throughout pregnancy.

Functional changes in the conformation of thrombospondin-1 during complexation with fibronectin or heparin.

Thrombospondin-1 (TSP-1) interacts specifically with heparin and fibronectin in vitro and colocalizes with fibronectin and heparan sulfate in the extracellular matrix (ECM). Its conformation is strongly dependent on Ca2+ concentration. We have previously shown that both heparin and fibronectin have two binding sites on the TSP-1 subunit which may require conformational change for their occupancy (R. Dardik and J. Lahav, 1987, Eur. J. Biochem. 168, 347; ibid 1989, 185, 581). To investigate the effect of TSP-1 binding to fibronectin and heparin on its functional conformation, TSP-1 was subjected to proteolysis in the presence and absence of ligands and of Ca2+. We found that while trypsin cleavage of free TSP-1 resulted in the inactivation of ligand binding, TSP-1 bound to either fibronectin or heparin remained stably associated with these ligands. Cleavage by thrombin or tissue plasminogen activator (tPA) showed that Ca2+-depleted TSP-1, when bound to fibronectin or to heparin, yielded proteolytic cleavage patterns typical of the Ca2+-containing form. Cleavage by chymotrypsin was not affected by binding to fibronectin or heparin; hence loss of proteolytic susceptibility was not due to steric hindrance by the ligands. Taken together, these results indicate that: (A) binding of TSP-1 to fibronectin or heparin is a two-step mechanism where binding to one site leads to conformational changes that enable binding to the second site; (B) TSP-1 in complex with fibronectin or heparin adopts the Ca2+-containing conformation in the absence of Ca2+; and (C) such complexes are highly resistant to cleavage by tPA and, if cleaved by other enzymes, the TSP-1 fragments remain bound to other ECM components. These characteristics have profound significance for platelet adhesion and cell migration into wounds where Ca2+ concentrations are reduced.

Cutaneous necrosis as a terminal paraneoplastic thromboembolic event in a patient with non-Hodgkin's lymphoma.

Thrombotic complications in non-Hodgkin's lymphoma often originate in the large veins. We describe a patient with refractory advanced high-grade lymphoma who presented with the rare complication of extensive cutaneous necrosis due to thrombosis of dermal vessels; there was also a recent new peak of monoclonal IgM-kappa protein. Direct immunofluorescence demonstrated immune deposits with complement in the dermal vessel wall. Based on these observations and on published data, we suggest that these complexes were the trigger for the thrombotic events and that the monoclonal IgM acted as xenoreactive antibodies, initiating a cascade of events. The first step of this cascade was activation of the complement and the membrane attack complex, which caused secretion of IL-1 alpha by endothelial cells, followed by overexpression of tissue factor on the surface of the dermal vessel wall endothelium. Dermal vessel thrombosis was the final event in this cascade.

Increased operative bleeding during orthopaedic surgery in patients with type I Gaucher disease and bone involvement.

To aid clinicians in identifying patients with type I Gaucher disease who are at risk of excessive bleeding, we reviewed the coagulation parameters of six affected patients with bone involvement who underwent orthopaedic surgery at two centers, and of 22 patients under treatment at another, seven of whom had total splenectomy. All patients were of Jewish Ashkenazi origin. Among the latter group, prolonged prothrombin time was noted in 81%. Incidence of clotting factor deficiency were as follows: factor XI, 36.3%; V, 31.8%; VIII, 27.2%; IX, 13.6%; and XII, 27.2%. Most of the abnormalities occurred in the non-splenectomized patients. Two of the six orthopaedic surgery patients had excessive intraoperative and postoperative bleeding. One, who underwent spinal decompression had prolonged prothrombin time, and the other, who had total hip replacement, showed a deficiency of factor XI. The second patient's hemoglobin level was maintained with transfusion of fresh frozen plasma during contralateral hip arthroplasty five months later. We suggest that preoperative evaluation of clotting factors and replacement therapy may prevent excessive bleeding in patients with type I Gaucher disease.

Activated protein C resistance in pediatric inflammatory bowel disease.

BACKGROUND: There is evidence for a hypercoagulable state in inflammatory bowel disease (IBD), and small vessel thrombosis has been identified in the bowel of patients with Crohn's disease, suggesting thrombosis as a possible etiologic factor. Activated protein C (APC) resistance is the most common inherited disorder leading to thrombosis and accounts for 30% to 40% of episodes of idiopathic venous thrombosis. METHODS: The prevalence of APC resistance was studied in 23 patients with IBD (17 with Crohn's disease, 6 with ulcerative colitis) and in 11 control subjects with recurrent abdominal pain or celiac disease, using an APC resistance screening method. RESULTS: One patient with Crohn's disease had a positive screen result, two patients (one with Crohn's, one with ulcerative colitis) had borderline results, and results in all of the control subjects were normal. One patient with Crohn's disease had a history of a thromboembolic event but had a normal screen result. CONCLUSIONS: Activated protein C resistance does not seem to play a major role in the etiology of the hypercoagulable state in inflammatory bowel disease.

Collagen binding induces changes in its platelet integrin receptor alpha2beta1.

Integrins can signal upon binding of their ligand, presumably because of conformational changes induced by ligand-binding. It has been postulated that ligand binding causes changes in the affinity of the integrin to its ligand. In order to test for ligand-induced change in the affinity of platelet alpha2beta1 to collagen, labelled viable platelets were passaged through a column of fibrillar collagen and stringent lysis conditions were used to remove all low-affinity receptors. A high-affinity fraction left on the collagen could be eluted with dithiothreitol (DTT) and 2% Sodium dodecyl sulfate (SDS). Antibodies raised against this fraction, identified alpha2beta1 by Western-blotting. Functional tests performed with the antibodies confirmed the involvement of the high-affinity proteins in platelet-collagen interactions attributed to alpha2beta1: inhibition of collagen-specific platelet adhesion and aggregation. EDTA, chaotropic agents or low pH did not elute the high affinity fraction of alpha2beta1. However, DTT followed by acetic acid did, which indicates that the steps necessary to disrupt the high-affinity collagen alpha2beta1 bond are reduction of disulfide bond(s) followed by disruption of electrostatic interactions. Our data 2 1 suggest that (i) ligand binding induces the formation of a new disulfide bond in a fraction of alpha2beta1, (ii) that this bond is an intrareceptor, and (iii) that this change increases the affinity of the receptor to its ligand.

Levels of proteins C and S do not decline subsequent to first line chemotherapy in lymphoma patients.

Thromboembolic complications and decrease in protein C and S have been observed in patients while receiving combination chemotherapy for breast cancer. We investigated whether initial cytotoxic treatment of non-Hodgkin's lymphoma (NHL) and Hodgkin's disease (HD) is also associated with changes in these anticoagulant parameters. For this purpose 25 patients with intermediate to high grade NHL and seven with HD, undergoing primary treatment with cytotoxic drugs were evaluated at three time-points: pre-therapy, mid-therapy and post-therapy. In contrast to the breast cancer patients, no significant changes in protein C, protein S and antithrombin III levels were observed in the NHL patients during the various stages of therapy. However in HD patients, the mean protein C values had a tendency to be higher at mid-therapy compared to pre-therapy and protein S levels had a tendency to be higher at mid-therapy compared to post-therapy. In lymphoma patients receiving primary cytotoxic treatment we did not find changes in anticoagulant parameters that can explain a chemotherapy-induced hypercoagulable state, as has been reported in breast cancer patients.

Affinity modulation in platelet alpha 2 beta 1 following ligand binding.

In order to test for ligand-induced change in the affinity of platelet alpha 2 beta 1 to collagen we passaged labeled viable platelets through a column of fibrillar collagen and used stringent lysis conditions to remove all low-affinity receptors. A high affinity fraction left on the collagen could be eluted with DTT and 2% SDS. Antibodies raised against it Western-blotted alpha 2 beta 1. Functional tests performed with the antibodies confirmed the involvement of the high affinity proteins in platelet-collagen interactions attributed to alpha 2 beta 1: inhibition of collagen-specific platelet adhesion and aggregation. EDTA, chaotropic agents or low pH did not elute the high affinity fraction of alpha 2 beta 1. However, DTT followed by acetic acid did. Our data suggest that 1) ligand binding induces the formation of a new disulfide bond in a fraction of alpha 2 beta 1, 2) that this bond is intrareceptor and 3) that this change increases the affinity of the receptor to its ligand.

A human monoclonal IgA autoantibody--185/12--behaves like an autoimmune antiphospholipid antibody.

A human monoclonal anticardiolipin autoantibody (ACA) of the IgA-k isotype, designated 185/12, is described. The antibody was prepared from peripheral B cells, obtained from a patient with a history of habitual abortion, by immortalization with Epstein-Barr virus (EBV). The antibody displays a strong binding activity to cardiolipin and phosphatidyl L-serine, but not to phosphatidylcholine, phosphatidylinositol, ssDNA and dsDNA. It binds to cardiolipin in a concentration-related and saturable manner (Kd = 3.0 x 10(-8) M). This reaction is dependent upon the presence of bovine serum, and is fully inhibited by cardiolipin vesicles. The 185/12 antibody exhibits different binding patterns to the solid-phase bound cardiolipin-serum complex and to its individual components (cardiolipin and bovine serum). The Bmax of 185/12 binding to the complex (0.968 OD units) is higher than the sum of the Bmax values calculated for each one of the complex components (0.352 + 0.179 = 0.531 OD units). Bovine serum as well as purified beta 2-glycoprotein I (beta 2-GPI) in suspension inhibit the binding of 185/12 to the complex. 185/12 binding capacity increases in direct relation to the rising concentration of beta 2-GPI. Collectively, these data may be interpreted to suggest that 185/12 antibody, which is an IgA isotype, exhibits characteristics usually attributed only to antiphospholipid autoantibodies (APA) of the IgG isotype, that are associated with the clinical spectrum of APA syndrome (APA-S). It is, therefore, possible that autoantibodies of the IgA isotype could play a pathogenic role, which may be different from that of the IgG isotype, in the development of autoimmune phenomena.

The functions of thrombospondin and its involvement in physiology and pathophysiology.

The thrombospondin family of molecules is expressed in many different tissues. Its expression is highly regulated by different hormones and cytokines and is developmentally controlled. It can bind to many different cell types, probably via an array of receptors which are similarly regulated. The level of thrombospondins in body fluids and their distribution in tissue change in correlation with various pathological states. It is linked to the growth of primary tumors and to metastasis, to development of the atherosclerotic plaque, to malaria infection and other diseases. The role(s) of thrombospondin(s) are by and large unknown, though specific interaction seem to affect particular cell functions. The wide-spread spatial and temporal regulation, multiple interactions and correlation with major diseases imply important roles in cell function and call for concerted effort to unravel the mystery.

Lupus anticoagulant antibodies inhibit collagen-induced adhesion and aggregation of human platelets in vitro.

The effect of circulating lupus anticoagulant on platelet interaction with collagen and other proteins was tested, with the aim of understanding the role of membrane phospholipids in platelet function. Plasma samples from 26 systemic lupus erythematosus (SLE) patients, containing circulating lupus anticoagulant (LAC), were examined for their effect on adhesion and aggregation of normal human platelets. We find that SLE plasma, but not normal plasma, inhibits platelet adhesion to collagen in a concentration-dependent manner. At a plasma concentration of 1% the inhibition was 73 +/- 9% (mean +/- SD). In sharp contrast, there was no effect on platelet adhesion to fibronectin. Purified IgG from the same plasma samples also had an inhibitory effect. At 15 micrograms/ml (comparable in IgG concentration to 0.1% plasma) it inhibited adhesion to collagen by 33 +/- 11%. Inhibition could be abolished by preincubation of the LAC-containing plasma with cardiolipin (CL), phosphatidylinositol (PI), and, to a lesser extent, phosphatidylserine (PS) but not with phosphatidylcholine (PC) or phosphatidylethanolamine (PE). Inhibition could also be abolished by preincubation of the LAC-containing plasma with a 10-fold excess of washed normal platelets. The effect of 1% LAC plasma on platelet aggregation was as striking, showing 79 +/- 26% inhibition of collagen-induced aggregation, and it could also be abolished by preincubation of the LAC plasma with cardiolipin. In contrast, the effect of LAC plasma on thrombin-induced aggregation was rather modest. Our results indicate that antiphospholipid antibodies interfere with platelet adhesion and stimulation by collagen in vitro and point to an important role of external plasma membrane phospholipids, particularly PI, in collagen-induced platelet activation.