Networks of enzymatically oxidized membrane lipids support calcium-dependent coagulation factor binding to maintain hemostasis.

Blood coagulation functions as part of the innate immune system by preventing bacterial invasion, and it is critical to stopping blood loss (hemostasis). Coagulation involves the external membrane surface of activated platelets and leukocytes. Using lipidomic, genetic, biochemical, and mathematical modeling approaches, we found that enzymatically oxidized phospholipids (eoxPLs) generated by the activity of leukocyte or platelet lipoxygenases (LOXs) were required for normal hemostasis and promoted coagulation factor activities in a Ca2+- and phosphatidylserine (PS)-dependent manner. In wild-type mice, hydroxyeicosatetraenoic acid-phospholipids (HETE-PLs) enhanced coagulation and restored normal hemostasis in clotting-deficient animals genetically lacking p12-LOX or 12/15-LOX activity. Murine platelets generated 22 eoxPL species, all of which were missing in the absence of p12-LOX. Humans with the thrombotic disorder antiphospholipid syndrome (APS) had statistically significantly increased HETE-PLs in platelets and leukocytes, as well as greater HETE-PL immunoreactivity, than healthy controls. HETE-PLs enhanced membrane binding of the serum protein ß2GP1 (ß2-glycoprotein 1), an event considered central to the autoimmune reactivity responsible for APS symptoms. Correlation network analysis of 47 platelet eoxPL species in platelets from APS and control subjects identified their enzymatic origin and revealed a complex network of regulation, with the abundance of 31 p12-LOX-derived eoxPL molecules substantially increased in APS. In summary, circulating blood cells generate networks of eoxPL molecules, including HETE-PLs, which change membrane properties to enhance blood coagulation and contribute to the excessive clotting and immunoreactivity of patients with APS.

Co-existence of renovascular hypertension, polyarteritis nodosa, antiphospholipid syndrome and methylenetetrahydrofolate reductase mutation.

We present a hypertensive child with a co-existence of polyarteritis nodosa, anti-phospholipid antibodies (aPL), methylenetetrahydrofolate reductase (MTHFR) mutation and increased lipoprotein a level. Elevated renin, aldosterone and aPL levels, micro-aneurysms, occlusion and thrombosis at left and right renal artery were found. Anti-hypertensive agents, prednisolone and pulse cyclophosphamide therapy were started and a stent was inserted in the left renal artery. Two months later, brain magnetic resonance imaging/magnetic resonance imaging angiography showed acute infarct area of the left parietofrontal lobe and middle cerebral artery stenosis. We found bilateral peripheral neuropathy, persistent aPL and elevated Lp(a) level and heterozygous A1298C/MTHFR mutation. Intravenous immunoglobulin and low-molecular-weight heparin treatment was added. In conclusion, our observation suggests that in patients with systemic vasculitis, such as polyarteritis nodosa, aPL are probably associated with greater thrombotic risks. The investigation of the LP(a) levels and MTHFR mutations as a synergic pro-coagulant effect might also be considered for determining patients with vasculitis at risk for severe thrombotic events.

Phospholipid scramblase 1 expression is enhanced in patients with antiphospholipid syndrome.

OBJECTIVE: Thrombus formation is the key event of vascular manifestations in antiphospholipid syndrome (APS). Phosphatidylserine (PS) is normally sequestered in the inner leaflet of cell membranes. Externalization of PS occurs during cell activation and is essential for promoting blood coagulation and for the binding of antiphospholipid antibodies (aPL) to cells. One of the molecules involved in PS externalization is phospholipid scramblase 1 (PLSCR1). We evaluated PLSCR1 expression on monocytes from APS patients and analyzed the in vitro effect of monoclonal aPL on PLSCR1 expression. PATIENTS AND METHODS: Forty patients with APS were investigated. In vitro experiments were performed in monocyte cell lines incubated with monoclonal aPL. PLSCR1 expression was determined by quantitative real-time polymerase chain reactions. PS exposure on CD14(+) cell surface was analyzed by flow cytometry. RESULTS: Levels of full-length PLSCR1 messenger RNA (mRNA) were significantly increased in APS patients compared with healthy controls (2.4 ± 1.2 vs. 1.3 ± 0.4, respectively, p < 0.001). In cultured monocytes, interferon alpha enhanced tissue-factor expression mediated by ß2-glycoprotein-I-dependent monoclonal anticardiolipin antibody. CONCLUSIONS: Monocytes in APS patients had increased PLSCR1 mRNA expression.

Factor XI is a substrate for oxidoreductases: enhanced activation of reduced FXI and its role in antiphospholipid syndrome thrombosis.

Factor XI (FXI), a disulfide-linked covalent homodimer, circulates in plasma, and upon activation initiates the intrinsic/consolidation phase of coagulation. We present evidence that disulfide bonds in FXI are reduced to free thiols by oxidoreductases thioredoxin-1 (TRX-1) and protein disulfide isomerase (PDI). We identified that Cys362-Cys482 and Cys118-Cys147 disulfide bonds are reduced by TRX-1. The activation of TRX-1-treated FXI by thrombin, FXIIa or FXIa was significantly increased compared to non-reduced FXI, indicating that the reduced factor is more efficiently activated than the oxidized protein. Using a novel ELISA system, we compared the amount of reduced FXI in antiphospholipid syndrome (APS) thrombosis patients with levels in healthy controls, and found that APS patients have higher levels of reduced FXI. This may have implication for understanding the contribution of FXI to APS thrombosis, and the predisposition to thrombosis in patients with elevated plasma levels of reduced FXI.

Pathophysiology of thrombosis and potential targeted therapies in antiphospholipid syndrome.

The antiphospholipid syndrome (APS) is an autoimmune disease in which recurrent vascular thrombosis, pregnancy morbidity or a combination of these events is associated with the persistent presence of circulating antiphospholipid antibodies (aPL). Evidence shows that the dominant antigenic targets for aPL in APS are phospholipid-binding plasma proteins such as b2glycoprotein I and prothrombin. The pathogenic role of aPL in thrombosis is widely accepted but the mechanisms by which these antibodies mediate disease are only partially understood. aPL may affect the normal procoagulant and anticoagulant reactions occurring on cell surface, and also may interact with certain cells, altering the expression and secretion of procoagulant substances. The intracellular signal transduction triggered by aPL has been a focus of intensive research and the p38 mitogen activated protein kinase (MAPK) pathway has been revealed as a major player in the aPL-mediated cell activation. In addition, some candidates as cell-receptor for phospholipid-binding plasma proteins have been identified. The recognition of the intracellular signaling triggered by aPL is a step forward in the design of new modalities of targeted therapies for thrombosis in APS including specific inhibitors of MAPK pathway or antagonists of the putative receptors. Furthermore, novel findings regarding the role of aPL in T-cells responses mark new advances in the understanding of the immunological reactions in APS and open new insights into possible therapeutic approaches to APS. In this article, we review the pathophysiological mechanisms of thrombosis and the specific new targeted therapies for the treatment in APS.

ß2-Glycoprotein-I based peptide regulate endothelial-cells tissue-factor expression via negative regulation of pGSK3ß expression and reduces experimental-antiphospholipid-syndrome.

Antiphospholipid syndrome (APS) is characterized by thromboembolic phenomena and recurrent fetal loss associated with elevated circulating anti-phospholipid/beta2glycoprotein-I(ß2GPI)-binding-antibodies(Abs). Individual APS patients harbor diverse clusters of circulating anti-ß2GPI Abs, targeting different epitopes on the ß2GPI molecule. Our novel approach was to construct a peptide composed of ß2GPI-ECs-binding-site (phospholipids-membrane), named "EMBI". EMBI exert dual activities: a) At first EMBI prevented ß2GPI ECs binding, thus reduced by 89% the binding of ß2GPI/anti-ß2GPI to the cells in comparison with 9.3% inhibition by EMBI scrambled form (scEMBI). b) Longer exposure of ECs to EMBI resulted in intracellular EMBI penetration which did not prevent ß2GPI/anti-ß2GPI binding to HUVEC. Surprisingly, ß2GPI/anti-ß2GPI did not activate ECs harboring EMBI, illustrated by prevention of E-selectin and tissue factor (TF) expression. The inhibition of TF mRNA transcription was illustrated by quantitative RT-PCR. EMBI decreased the expression of phosphorylated JNK1/2, p38, HSP27 and enhanced phosphorylation of glycogen synthase kinase-3ß (pGSK3ß). Knocking down the GSK3ß expression by siRNA-GSK3ß, reduced the TF expression by ß2GPI/anti-ß2GPI-exposed-HUVEC. In-vivo, EMBI significantly decreased the percentage of fetal loss in naïve mice infused with anti-ß2GPI Abs, p<0.04. Thus, the dual activity of EMBI may introduce EMBI as a potential novel candidate peptide, to treat patients with APS.

Increased expression of phospholipid scramblase 1 in monocytes from patients with systemic lupus erythematosus.

OBJECTIVE: A high incidence of thromboembolic events has been reported in patients with systemic lupus erythematosus (SLE). Phosphatidylserine (PS) is normally sequestered in the inner leaflet of cell membranes. Externalization of PS during cell activation is mediated by phospholipid scramblase 1 (PLSCR1) and has a central role in promoting blood coagulation. We investigated the underlying pathogenic status of thrombophilia in SLE by analyzing PLSCR1 expression on monocytes from patients with SLE. METHODS: Sixty patients with SLE were evaluated. Twenty-three patients had antiphospholipid syndrome (APS/SLE). Plasma D-dimer levels were measured as a marker of fibrin turnover. The cDNA encoding human PLSCR1 was cloned from the total RNA extract from monocytes, and independent clones were sequenced. PLSCR1 mRNA expression in CD14+ cells was determined by real-time polymerase chain reaction. PS exposure on CD14+ cell surface was analyzed by flow cytometry. RESULTS: Elevated D-dimer levels were found in plasma from SLE patients. Three splice variants of PLSCR1 mRNA were identified in all subjects, and levels of full-length PLSCR1 mRNA were significantly increased in SLE compared to healthy controls (2.9 +/- 1.5 vs 1.3 +/- 0.4, respectively; p < 0.0001). Flow-cytometry analysis showed relative enhancement of PS exposure in the surface of CD14+ cells in SLE patients compared to healthy controls. CONCLUSION: Novel PLSCR1 splice variants were identified. Monocytes in SLE patients had enhanced PLSCR1 mRNA expression, as well as increased fibrin turnover and cell-surface PS exposure, indicating that PLSCR1 may, in part, contribute to the prothrombotic tendency in SLE.

Identification of anti-prothrombin antibodies in the anti-phospholipid syndrome that display the prothrombinase activity.

OBJECTIVE: Prothrombin (PT) is one of the most important antigenic targets for aPL antibodies; however, the prothrombotic mechanism of anti-PT (aPT) antibodies in APS is not fully clarified. Considering that some autoantibodies possess the enzymatic activity, the aim of this study was to test the hypothesis that some aPT antibodies in APS may display prothrombinase activity. METHODS: Six APS patient-derived PT-reactive monoclonal antibodies (mAbs) were analysed for prothrombinase activity on PT. One mAb with prothrombinase activity was examined for its proteolytic activity on PT. In addition, IgG was purified from plasma samples positive with IgG aPT antibodies, and their prothrombinase activity analysed. RESULTS: Initial analysis of six mAbs revealed that, upon incubation with PT, IS6 mAb displayed prothrombinase activity and catalysed the proteolysis of PT to fragments. Analysis of plasma samples revealed that 9/21 (42.8%) APS patients had IgG antibodies against PT, based on a cut-off value equal to mean + 3 S.D. of the level in 21 normal controls. Importantly, of those samples positive for IgG aPT antibodies, two polyclonal IgG (P1 and P2) also displayed prothrombinase activity. CONCLUSIONS: In this study, we showed that some aPT antibodies displayed prothrombinase activity. Such catalytic aPT antibodies may contribute to thrombosis in APS.

Vascular abnormalities, paraoxonase activity, and dysfunctional HDL in primary antiphospholipid syndrome.

CONTEXT: Accelerated atherosclerosis has been described in antiphospholipid syndrome, but the vascular abnormalities and the underlying mechanisms remain unclear. OBJECTIVES: To compare vascular structure and function in patients with positive antiphospholipid antibodies (aPL) with controls and to assess their relationship with paraoxonase activity. DESIGN, SETTING, AND PARTICIPANTS: A cross-sectional study of 77 women with positive antiphospholipid antibodies from a lupus outpatient clinic in London, England (90% of the eligible population) and 77 controls matched on frequency basis for age and cardiovascular risk factors between June 2006 and April 2009. Carotid intima media thickness (CIMT), flow-mediated dilatation, pulse wave velocity, and paraoxonase activity were measured in all patients. Anti-inflammatory and antioxidant properties of high-density lipoprotein (HDL) were examined. MAIN OUTCOME MEASURES: CIMT, pulse wave velocity, flow-mediated dilatation, and paraoxonase. RESULTS: Women with aPL had greater CIMT and pulse wave velocity compared with controls (mean [SD], 0.75 [0.16] vs 0.64 [0.09] mm; 95% confidence interval [CI], -0.14 to -0.06; P < .001; and 9.2 [1.6] vs 8.5 [1.8] m/s; 95% CI, -1.14 to -0.06; P = .04) and lower flow-mediated dilatation (6.2% [4.1%] vs 9.6% [4.2%]; 95% CI, 2.02%-4.69%; P < .001). Paraoxonase activity was lower in women with aPL vs controls (median [interquartile range], 91.2 [64.3-105.1] vs 103.0 [80.5-111.5] micromol p-nitrophenol/L/serum/min; 95% CI, 0.004-0.007; P = .005) and was inversely associated with CIMT and pulse wave velocity in women with aPL (standardized beta coefficient = -0.4 and -0.3, respectively; P < .05 for both), but not in the control group. High-density lipoprotein from women with aPL inhibited endothelial nitric oxide production in human aortic endothelial cells, in contrast with controls. The beneficial effects of HDL from women with aPL on vascular cell adhesion molecule 1 expression, superoxide production, and monocyte adhesion following activation of human aortic endothelial cells were largely blunted. CONCLUSIONS: Compared with controls, women with aPL had greater functional and structural arterial abnormalities, which were associated with lower activity of paraoxonase. In patients with aPL, HDL reduced nitric oxide bioavailability and had impaired anti-inflammatory and antioxidant properties.

Identification of anti-plasmin antibodies in the antiphospholipid syndrome that inhibit degradation of fibrin.

The combined presence of anti-phospholipid Ab (aPL) and thrombosis is recognized as the antiphospholipid syndrome (APS). The aPL represent a heterogeneous group of Ab that recognize various phospholipids (PL), PL-binding plasma proteins, and/or PL-protein complexes. Recently, we found the presence of antithrombin Ab in some APS patients and that some of these anti-thrombin Ab could inhibit thrombin inactivation by antithrombin. Considering that thrombin is homologous to plasmin, which dissolves fibrin, we hypothesize that some APS patients may have Ab that react with plasmin, and that some anti-plasmin Ab may interfere with the plasmin-mediated lysis of fibrin clots. To test this hypothesis, we searched for anti-plasmin Ab in APS patients and then studied those found for their effects on the fibrinolytic pathway. The results revealed that seven of 25 (28%) APS patients have IgG anti-plasmin Ab (using the mean OD plus 3 SD of 20 normal controls as the cutoff) and that six of six patient-derived IgG anti-thrombin mAb bind to plasmin with relative K(d) values ranging from 5.6 x 10(-8) to 1 x 10(-6) M. These K(d) values probably represent affinities in the higher ranges known for human IgG autoantibodies against protein autoantigens. Of these mAb, one could reduce the plasmin-mediated lysis of fibrin clots. These findings suggest that plasmin may be an important driving Ag for some aPL B cells in APS patients, and that the induced anti-plasmin Ab may act either directly, by binding to plasmin and inhibiting its fibrinolytic activity, or indirectly, by cross-reacting with other homologous proteins in the coagulation cascade to promote thrombosis.

[Mutation of a 5,10-methylenetetrahydrofolate reductase gene in systemic lupus erythematosis and antiphospholipid syndrome]. / Mutatsiia v gene 5,10-metilentetragidrofolatreduktazy pri sistemnoi krasnoi volchanke i antifosfolipidnom sindrome.

AIM: To study prevalence of mutation C677T in gene 5.10-MTHFR in systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS) as well as in persons free of symptoms of systemic diseases of the connective tissue. MATERIAL AND METHODS: 85 patients participating in the study were divided into three groups: those with SLE (n = 17), with SLE + APS (n = 42), with primary APS (n = 26). The control group consisted of 30 persons without SLE or APS. 55% of the examinees had thrombotic complications of different location. The diagnosis of the mutation was made using DNA isolated from the peripheral blood with standard methods and polymerase chain reaction. Allele (homozygous or heterozygous) condition of the mutation was confirmed by means of allele-specific primers. RESULTS: Mutation C677T in MTHFR gene was found in 40 of 85 patients (47%); 11(27.5%) had a homozygous variant, 29(72.5%)--heterozygous. C677T mutation occurred in 5 of 17 SLE patients (29%), it was in all the cases heterozygous. In primary and secondary APS mutation was detected in 51.5% (35 of 68 patients). Recurrent thrombosis occurred more frequently in patients with mutation MTHFR. Three and more episodes of thrombosis were registered in 17 of 40 patients with mutation C677T against 9 of 44 patients without the mutations (p = 0.04). CONCLUSION: Relationship between elevated blood levels of APL and MTHFR mutation points to the fact that this genetic marker is an additional thrombogenic factor in APS. Mutation C677T in MTHFR gene in APS patients correlates with recurrent thrombosis.

Paraoxonase activity is dramatically decreased in patients positive for anticardiolipin antibodies.

It has been reported that paraoxonase 1 (PON1) activity inhibits low-density lipoprotein (LDL) oxidation and modulates the risk of coronary heart disease. This study shows that autoantibodies (IgG) directed against modified LDL were increased in 71 patients positive for anticardiolipin antibodies. In a representative subgroup of these patients (n = 36) PON1 activity was dramatically decreased and the prevalence of the RR genotype of this enzyme tended to be increased in patients who had developed arterial thrombosis. This study suggests that PON1 abnormalities play a role in the antiphospholipid syndrome.

[Biochemical characteristics of liver involvement in patients with antiphospholipid syndrome]. / Biokhimicheskie osobennosti porazheniia pecheni u bol'nykh s antifosfolipidnym sindromom.

The incidence of hepatitis B and C virus and cytomegalovirus infection is high in patients with the antiphospholipid syndrome (APS). The specific features of virus infection in APS patients are determined by the activity of APS. During clinically manifest stage, the activities of aminotransferases, lactate dehydrogenase (LDH), and alkaline phosphatase increase, while during remission only aspartate aminotransferase and LDH levels remain high, for this latter enzyme high activities of isoenzymes LDH5 and LDH4 being recorded. These data indicate that the pathological process in APS involves not only the liver, but the sinusoidal endothelium as well. This seems to account for some other clinical and laboratory manifestations of APS, such as increased level of circulating immune complexes, dysfunction of physiological anticoagulants, etc.

Decreased mRNA levels coding for poly(ADP-ribose) polymerase in lymphocytes of patients with SLE.

Poly(ADP-ribose) metabolism is altered in patients with SLE. In order to localize the defect, the levels of poly(ADP-ribose) polymerase-specific mRNA were measured from dot blots of total RNA from peripheral blood lymphocytes. In this preliminary study, eleven patients with SLE and two with antiphospholipid syndrome were compared to three controls. It was found that the mean levels of specific mRNA were ten fold lower in the PBL from SLE patients compared to controls and no overlap of values was seen between the two groups. No such decrease was seen in the PBL from the patients with antiphospholipid syndrome. It is concluded that the defect in poly(ADP-ribose) polymerase metabolism that is seen in SLE patients occurs at the level of transcription or mRNA turnover.