Kwan-Ki Hwang

Identification of Anti-Thrombin Antibodies in the Antiphospholipid Syndrome That Interfere with the Inactivation of Thrombin by Antithrombin

The combined presence of anti-phospholipid (PL) Ab, including lupus anticoagulants (LAC) and/or anticardiolipin Ab (aCL), and thrombosis is recognized as the antiphospholipid syndrome (APS). LAC are detected as an inhibitory effect on PL-restricted in vitro blood coagulation tests, and are comprised mainly of Ab against β2 glycoprotein I and prothrombin (PT). Recently, anti-PT Ab (aPT) were found to be associated with thrombosis by some investigators, although this is not confirmed by others. Considering that aPT are heterogeneous in patients and that PT is converted into thrombin, we hypothesize that certain aPT in patients may bind to thrombin, and that some of such anti-thrombin Ab may interfere with thrombin-antithrombin (AT) interaction and thus reduce the AT inactivation of thrombin. To test this hypothesis, we searched for anti-thrombin Ab in APS patients and then studied those found for their effects on the AT inactivation of thrombin. The results revealed that most, but not all, aPT-positive patient plasma samples contained anti-thrombin Ab. To study the functional significance of these Ab, we identified six patient-derived mAb that bound to both PT and thrombin. Of these mAb, three could reduce the AT inactivation of thrombin, whereas others had minimal effect. These findings indicate that some aPT in patients react with thrombin, and that some of such anti-thrombin Ab could inhibit feedback regulation of thrombin. Because the latter anti-thrombin Ab are likely to promote clotting, it will be important to develop specific assays for such Ab and study their roles in thrombosis in APS patients.

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 Kd values ranging from 5.6 × 10−8 to 1 × 10−6 M. These Kd 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.

Antibodies against the Activated Coagulation Factor X (FXa) in the Antiphospholipid Syndrome That Interfere with the FXa Inactivation by Antithrombin

Antiphospholipid Ab have been shown to promote thrombosis and fetal loss in the antiphospholipid syndrome (APS). Previously, we found IgG anti-thrombin Ab in some APS patients that could interfere with inactivation of thrombin by antithrombin (AT). Considering that activated coagulation factor X (FXa) is homologous to thrombin in the catalytic domains and is also regulated primarily by AT, we hypothesized that some thrombin-reactive Ab may bind to FXa and interfere with AT inactivation of FXa. To test these hypotheses, we studied reactivity of eight patient-derived monoclonal IgG antiphospholipid Ab with FXa and the presence of IgG anti-FXa Ab in APS patients and investigated the effects of FXa-reactive mAb on AT inactivation of FXa. The results revealed that six of six thrombin-reactive IgG mAb bound to FXa and that the levels of plasma IgG anti-FXa Ab in 38 APS patients were significantly higher than those in 30 normal controls (p < 0.001). When the mean plus 3 SDs of the 30 normal controls was used as the cutoff, 5 of 38 APS patients (13.2%) had IgG anti-FXa Ab. Importantly, three of six FXa-reactive mAb significantly inhibited AT inactivation of FXa. Combined, these results indicate that anti-FXa Ab may contribute to thrombosis by interfering with the anticoagulant function of AT on FXa in some APS patients.

Novel Autoantibodies against the Activated Coagulation Factor IX (FIXa) in the Antiphospholipid Syndrome That Interpose the FIXa Regulation by Antithrombin

We previously reported that some human antiphospholipid Abs (aPL) in patients with the antiphospholipid syndrome (APS) bind to the homologous enzymatic domains of thrombin and the activated coagulation factor X (FXa). Moreover, some of the reactive Abs are prothrombotic and interfere with inactivation of thrombin and FXa by antithrombin (AT). Considering the enzymatic domain of activated coagulation factor IX (FIXa) is homologous to those of thrombin and FXa, we hypothesized that some aPLs in APS bind to FIXa and hinder AT inactivation of FIXa. To test this hypothesis, we searched for IgG anti-FIXa Abs in APS patients. Once the concerned Abs were found, we studied the effects of the Ab on FIXa inactivation by AT. We found that 10 of 12 patient-derived monoclonal IgG aPLs bound to FIXa and that IgG anti-FIXa Abs in APS patients were significantly higher than those in normal controls (p < 0.0001). Using the mean + 3 SD of 30 normal controls as the cutoff, the IgG anti-FIXa Abs were present in 11 of 38 (28.9%) APS patients. Importantly, 4 of 10 FIXa-reactive monoclonal aPLs (including the B2 mAb generated against β2-glycoprotein I significantly hindered AT inactivation of FIXa. More importantly, IgG from two positive plasma samples were found to interfere with AT inactivation of FIXa. In conclusion, IgG anti-FIXa Ab occurred in ∼30% of APS patients and could interfere with AT inactivation of FIXa. Because FIXa is an upstream procoagulant factor, impaired AT regulation of FIXa might contribute more toward thrombosis than the dysregulation of the downstream FXa and thrombin.

Some Plasmin-Induced Antibodies Bind to Cardiolipin, Display Lupus Anticoagulant Activity and Induce Fetal Loss in Mice

The combined presence of anti-phospholipid Ab (aPL), thrombosis, and/or fetal loss is recognized as the antiphospholipid syndrome (APS). aPL include anti-cardiolipin Ab (aCL) and/or lupus anticoagulants (LAC, detected as Ig that prolong certain in vitro phospholipid (PL)-restricted blood clotting tests); both aCL and LAC are the diagnostic Ab for APS. Studies show that aPL represent a heterogeneous group of Ab, which recognize various PL, PL-binding plasma proteins, and/or PL-protein complexes. Recently, we found that five of seven patient-derived IgG monoclonal aCL react with thrombin, activated protein C, and plasmin. All three proteins are trypsin-like serine proteases (SP), and are highly homologous in their catalytic domains. Importantly, among these SP autoantigens, the reactive aCL bind to plasmin with the highest affinity, suggesting that plasmin may serve as a major driving autoantigen for some aCL in ∼30% of APS patients who are positive for IgG anti-plasmin Ab. To test this hypothesis, we immunized BALB/c mice with human plasmin and analyzed immune sera for aCL activity and reactivity with relevant SP. We found that some immune sera displayed aCL activity and/or bound to test SP. Subsequently, eight mAb were obtained and studied. The results revealed that one mAb displayed the aCL and the LAC activities and induced fetal loss when injected into pregnant mice. Immunohistological analyses of placentas revealed extensive deposits of activated C3 components. Combined, these data demonstrate that plasmin may serve as a driving Ag for some pathogenic aPL.

Plasmin immunization preferentially induces potentially prothrombotic IgG anticardiolipin antibodies in MRL/MpJ mice.

OBJECTIVE To test the hypothesis, utilizing 2 experimental mouse models, that plasmin is an important autoantigen that drives the production of certain IgG anticardiolipin (aCL) antibodies in patients with the antiphospholipid syndrome. METHODS BALB/cJ and MRL/MpJ mice were immunized with Freunds complete adjuvant in the presence or absence of human plasmin. The mouse sera were analyzed for production of IgG antiplasmin, IgG aCL, and IgG anti-beta(2)-glycoprotein I (anti-beta(2)GPI) antibodies. IgG monoclonal antibodies (mAb) were generated from the plasmin-immunized MRL/MpJ mice with high titers of aCL, and these 10 mAb were studied for their binding properties and functional activity in vitro. RESULTS Plasmin-immunized BALB/cJ mice produced high titers of IgG antiplasmin only, while plasmin-immunized MRL/MpJ mice produced high titers of IgG antiplasmin, IgG aCL, and IgG anti-beta(2)GPI. Both strains of mice immunized with the adjuvant alone did not develop IgG antiplasmin or IgG aCL. All 10 of the IgG mAb bound to human plasmin and cardiolipin, while 4 of 10 bound to beta(2)GPI, 3 of 10 bound to thrombin, and 4 of 10 bound to the activated coagulation factor X (FXa). Functionally, 4 of the 10 IgG mAb inhibited plasmin activity, 1 of 10 hindered inactivation of thrombin by antithrombin III, and 2 of 10 inhibited inactivation of FXa by antithrombin III. CONCLUSION Plasmin immunization leads to production of IgG antiplasmin, aCL, and anti-beta(2)GPI in MRL/MpJ mice, but leads to production of only IgG antiplasmin in BALB/cJ mice. IgG mAb generated from plasmin-immunized MRL/MpJ mice bind to various antigens and exhibit procoagulant activity in vitro. These results suggest that plasmin may drive potentially prothrombotic aCL in genetically susceptible individuals.