Anticoagulant Activity of β2-Glycoprotein I Is Potentiated by a Distinct Subgroup of Anticardiolipin Antibodies

1992 ◽  
Vol 68 (03) ◽  
pp. 297-300 ◽  
Author(s):  
Monica Galli ◽  
Paul Comfurius ◽  
Tiziano Barbui ◽  
Robert F A Zwaal ◽  
Edouard M Bevers
Keyword(s):  
Human Plasma ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Type A ◽  
Lipid Vesicles ◽  
Dependent Manner ◽  
Type B ◽  
Distinct Subgroup ◽  
Glycoprotein I ◽  
Thrombin Formation

SummaryPlasmas of 16 patients positive for both IgG anticardiolipin (aCL) antibodies and lupus anticoagulant (LA) antibodies were subjected to adsorption with liposomes containing cardiolipin. In 5 of these plasmas both the anticardiolipin and the anticoagulant activities were co-sedimented with the liposomes in a dose-dependent manner, whereas in the remaining cases only the anticardiolipin activity could be removed by the liposomes, leaving the anticoagulant activity (LA) in the supernatant plasma. aCL antibodies purified from the first 5 plasmas were defined as aCL-type A, while the term aCL-type B was used for antibodies in the other 11 plasmas, from which 2 were selected for this study.Prolongation of the dRVVT was produced by affinity-purified aCL-type A antibodies in plasma of human as well as animal (bovine, rat and goat) origin. aCL-type B antibodies were found to be devoid of anticoagulant activity, while the corresponding supernatants containing LA IgG produced prolongation of the dRVVT only in human plasma.These anticoagulant activities of aCL-type A and of LA IgG's were subsequently evaluated in human plasma depleted of β2-glycoprotein I (β2-GPI), a protein which was previously shown to be essential in the binding of aCL antibodies to anionic phospholipids. Prolongation of the dRVVT by aCL-type A antibodies was abolished using β2-GPI deficient plasma, but could be restored upon addition of β2-GPI. In contrast, LA IgG caused prolongation of the dRVVT irrespective of the presence or absence of β2-GPI.Since β2-GPI binds to negatively-charged phospholipids and impedes the conversion of prothrombin by the factor Xa/Va enzyme complex (Nimpf et al., Biochim Biophys Acta 1986; 884: 142–9), comparison was made of the effect of aCL-type A and aCL-type B antibodies on the rate of thrombin formation in the presence and absence of β2-GPI. This was measured in a system containing highly purified coagulation factors Xa, Va and prothrombin and lipid vesicles composed of 20 mole% phosphatidylserine and 80 mole% phosphatidylcholine. No inhibition on the rate of thrombin formation was observed with both types of aCL antibodies when either β2-GPI or the lipid vesicles were omitted. Addition of β2-GPI to the prothrombinase assay in the presence of lipid vesicles causes a time-dependent inhibition which was not affected by the presence of aCL-type B or non-specific IgG. In contrast, the presence of aCL-type A antibodies dramatically increased the anticoagulant effect of β2-GPI. These data indicate that the anticoagulant activity of aCL-type A antibodies in plasma is mediated by β2-GPI.

scholarly journals Kaolin clotting time and dilute Russell's viper venom time distinguish between prothrombin-dependent and beta 2-glycoprotein I-dependent antiphospholipid antibodies

Blood ◽  
1995 ◽  
Vol 86 (2) ◽  
pp. 617-623 ◽  
Author(s):  
M Galli ◽  
G Finazzi ◽  
EM Bevers ◽  
T Barbui
Keyword(s):  
Factor Xa ◽  
Type A ◽  
The Other ◽  
Type B ◽  
Clotting Time ◽  
Positive Group ◽  
Glycoprotein I ◽  
Factor Ixa ◽  
Beta 2 ◽  
Kaolin Clotting Time

Antiphospholipid (aPL) antibodies include anticardiolipin (aCL) and lupus anticoagulant (LA) antibodies. LA antibodies recognize the complex of lipid-bound (human) prothrombin, in this way inhibiting the phospholipid-dependent coagulation reactions, whereas aCL antibodies are directed towards beta 2-glycoprotein I (beta 2-GPI) bound to an anionic lipid surface. According to their behavior in coagulation reactions, we have divided aCL antibodies into two groups: aCL-type A, which inhibit the phospholipid-dependent coagulation reactions because they enhance the binding of beta 2-GPI to the procoagulant phospholipid surface; and aCL-type B antibodies, which are devoid of anticoagulant properties. We report the distinctive laboratory and clinical profiles of 25 patients with well-characterized, phospholipid-dependent inhibitor of coagulation. Fourteen patients had LA antibodies (aCL-type B were concomitantly present in 10 cases, while in the other four, aCL titer was normal), and the other 11 had aCL-type A antibodies. The laboratory evaluation of the two groups showed the dilute Russell viper venom time (dRVVT) to be the most abnormal coagulation test in the aCL- type A-positive group, whereas the kaolin clotting time (KCT) was the most abnormal assay in the LA-positive group. In fact, the ratios of the coagulation times of patient plasma over normal pooled plasma (mean +/- standard deviation) for LA versus aCL-type A antibodies were 1.48 +/- 0.27 versus 2.20 +/- 0.42, P = .0001, and 2.22 +/- 0.42 versus 1.50 +/- 0.42, P = .0003, for the dRVVT and KCT, respectively. No differences were observed either in the ratios of the activated partial thromboplastin times and the prothrombin times or the plasma levels of beta 2-GPI and prothrombin. Conversely, aCL titers were significantly higher in aCL-type A-positive patients (147 +/- 44 U) than in the LA- positive group (61 +/- 55 U; P = .0003). We ruled out the possibility that platelet contamination of plasma could account for the observed coagulation profiles, as the two patterns were reproduced in platelet- free plasma. In addition, we performed clotting tests in plasma in the presence of phospholipids and calcium after addition of factor IXa or factor Xa. The assay performed with factor Xa was more sensitive to the presence of aCL-type A antibodies, while the assay performed with factor IXa was preferentially sensitive to LA-containing plasmas, supporting the earlier findings with the dRVVT and KCT assays.(ABSTRACT TRUNCATED AT 400 WORDS)

IgG antibodies that recognize epitope Gly40-Arg43 in domain I of β2–glycoprotein I cause LAC, and their presence correlates strongly with thrombosis

Blood ◽  
2005 ◽  
Vol 105 (4) ◽  
pp. 1540-1545 ◽  
Author(s):  
Bas de Laat ◽  
Ronald H. W. M. Derksen ◽  
Rolf T. Urbanus ◽  
Philip G. de Groot
Keyword(s):  
Lupus Anticoagulant ◽  
Anticoagulant Activity ◽  
Type A ◽  
Full Length ◽  
Enzyme Linked Immunosorbent Assay ◽  
Igg Antibodies ◽  
Type B ◽  
Glycoprotein I ◽  
Heterogeneous Reactivity ◽  
Domain I

AbstractAnti–β2–glycoprotein I antibodies are known to have a heterogeneous reactivity against β2–glycoprotein I. We performed this study to characterize the epitope on β2–glycoprotein I to which pathologic anti–β2–glycoprotein I antibodies are directed. Plasma samples from 198 patients with various systemic autoimmune diseases were tested for the presence of lupus anticoagulant and anti–β2–glycoprotein I immunoglobulin G (IgG) antibodies. The reactivity of the anti–β2–glycoprotein I–positive samples was further tested by coating recombinant full-length β2–glycoprotein I and 8 deletion mutants of β2–glycoprotein I onto hydrophilic and hydrophobic enzyme-linked immunosorbent assay (ELISA) plates. Full-length β2–glycoprotein I with point mutations in domain I at positions 8, 40, and 43 were used in inhibition experiments. Fifty-two patients with anti–β2–glycoprotein I IgG antibodies could be divided into 2 patterns. Type A antibodies only recognize domain I when coated onto hydrophobic plates; they do not recognize domain I coated onto hydrophilic plates. Type B antibodies have heterogeneous reactivity for all domains. Type A antibodies recognize the epitope around amino acids Gly40-Arg43 and cause lupus anticoagulant activity. In contrast to type B antibodies, those of type A strongly correlated with thrombosis. In conclusion, antibodies directed at domain I (epitope comprising Gly40 and Arg43) have lupus anticoagulant activity and strongly associate with thrombosis.

In Vitro Evaluation of Apixaban, a Novel, Potent, Selective and Orally Bioavailable Factor Xa Inhibitor.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4130-4130 ◽  
Author(s):  
Joseph M. Luettgen ◽  
Tracy A. Bozarth ◽  
Jeffrey M. Bozarth ◽  
Frank A. Barbera ◽  
Patrick Y. Lam ◽  
...  
Keyword(s):  
Human Plasma ◽  
Serine Proteases ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Competitive Inhibitor ◽  
Rat Plasma ◽  
Reversible Inhibitor ◽  
Antithrombotic Agent

Abstract Apixaban, previously known as BMS-562247, is a high affinity, highly selective, orally-active, reversible inhibitor of coagulation factor Xa (fXa), in clinical studies as a therapeutic agent for prevention and treatment of thromboembolic diseases. The in vitro characteristics of apixaban were evaluated in purified systems and in human blood from healthy volunteers. Detailed kinetic analysis of apixaban inhibition of human fXa showed that it is a readily reversible, potent and competitive inhibitor versus a synthetic tripeptide substrate with a Ki of 0.08 nM, an association rate of 2 × 107 M−1s−1and a dissociation half life of 3.4 min. Weak affinity (Ki ~3 μM) is observed for thrombin, plasma kallikrein, and chymotrypsin. Affinity for trypsin and all other serine proteases tested is negligible with Ki > 15 μM. Apixaban is an effective inhibitor of free fXa and of prothrombinase, in buffer, platelet poor plasma, and whole blood. The anticoagulant activity of apixaban was determined in platelet-poor human plasma. Apixaban causes concentration dependent prolongation of the fXa mediated clotting assays. The human plasma concentration required to produce a doubling of the clotting time is 3.6 μM for prothrombin time, 7.4 μM for activated partial thromboplastin time and 0.4 μM for HepTest. To support preclinical efficacy and safety studies purified fXa from rabbit, dog and rat plasma was also found to be inhibited by apixaban (0.17, 2.6, and 1.3 nM, respectively). In summary the in vitro properties of apixaban show that it is a highly selective and potentially potent antithrombotic agent for venous and arterial thrombotic diseases.

Expression and Assembly of Procoagulant Complexes by Human Pleural Mesothelial Cells

1994 ◽  
Vol 71 (05) ◽  
pp. 587-592 ◽  
Author(s):  
Anuradha Kumar ◽  
Kathleen B Koenig ◽  
Alice R Johnson ◽  
Steven Idell
Keyword(s):  
Tissue Factor ◽  
Factor Xa ◽  
Mesothelial Cells ◽  
Factor Vii ◽  
Dependent Manner ◽  
Factor X ◽  
Specific Concentration ◽  
Pleural Mesothelial Cells ◽  
Direct Binding ◽  
Thrombin Formation

SummaryMany pleural diseases involve fibrin deposition within the pleural cavity, an event that necessarily involves the mesothelium. This study of human pleural mesothelial cells (HPMC) was designed to determine how the mesothelium initiates and sustains the coagulation process. We used functional assays for activation of both factor X and prothrombin to examine expression and assembly of procoagulant activity by human pleural mesothelial cells in culture. The rates of factor Xa and thrombin formation were calcium-dependent. The rate of factor Xa formation in the presence of added factor VII increased in a concentration-dependent manner, suggesting that tissue factor is the primary procoagulant associated with HPMC. The fact that direct binding of radioiodinated factor Vila to HPMC was specific, concentration-dependent and saturable confirms that tissue factor is expressed on the cell surface. The rate of thrombin formation increased with factor Xa concentration, and the rate was 5-, 6-fold higher in presence of added factor Va indicating that HPMC support expression of prothrombinase activity. Further, direct binding of radioiodinated factor Xa to HPMC was specific, concentration-dependent and saturable, confirming that the cells support the assembly of the prothrombinase complex.

Direct Thrombin Inhibitors, but Not Factor Xa Inhibitors, Enhance Thrombin Formation in Human Plasma by Interfering with the Thrombin–Thrombomodulin–Protein C System

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 528-528 ◽  
Author(s):  
Elisabeth Perzborn ◽  
Michaela Harwardt
Keyword(s):  
Protein C ◽  
Thrombin Generation ◽  
Factor Xa ◽  
Lag Time ◽  
Thrombin Inhibitors ◽  
Direct Thrombin Inhibitors ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Normal Plasma ◽  
Thrombin Formation

Abstract Activation of protein C is dependent on thrombin complexed with thrombomodulin (TM). Activated protein C (APC), together with its cofactor protein S, degrades coagulation Factors Va and VIIIa, thereby limiting further thrombin formation. Thus, in addition to suppressing the procoagulant effects of thrombin, direct thrombin inhibitors (DTIs) may also downregulate anticoagulant effects of thrombin-mediated feedback mechanisms. By contrast, direct Factor Xa (FXa) inhibitors block the formation of thrombin, but not its actions. The objective of this study was to investigate whether the direct FXa inhibitor, rivaroxaban, and the DTIs, dabigatran and melagatran, inhibit the negative-feedback reaction of the thrombin–TM complex/APC (thrombin–TM/APC) system and thereby increase thrombin formation. Experiments were conducted in plateletpoor plasma from healthy donors (normal plasma) and in pooled protein C-deficient plasma, both substituted with 1.33 μM phospholipids, in the presence or absence of 10 nM TM with increasing concentrations of rivaroxaban, dabigatran, melagatran, or the appropriate vehicles. Thrombin formation was initiated by adding 1.67 pM tissue factor (TF) and assessed by measuring the cleavage of the fluorogenic substrate Z-Gly-Gly-Arg-AMC (Bachem) using the Calibrated Automated Thrombogram (CAT, Thrombinoscope® BV) method. The parameters assessed were lag time, time to peak thrombin generation (tmax), peak thrombin generation (Cmax), and endogenous thrombin potential (ETP). In addition, formation of prothrombin fragments 1+2 (F1+2) was determined by ELISA (Enzygnost® F1+2 monoclonal [Dade Behring]). Rivaroxaban potently inhibited thrombin formation in the absence and presence of TM across all parameters in a concentration-dependent manner in both normal plasma and protein C-deficient plasma (see Table). In the absence of TM, melagatran and dabigatran also inhibited thrombin formation in a concentration-dependent manner, both in normal plasma and protein C-deficient plasma. In the presence of TM, DTIs prolonged lag time and tmax in a concentration-dependent manner. However, only high concentrations of the DTIs reduced ETP, Cmax, and F1+2, In normal plasma,lower concentrations even increased ETP, Cmax, and F1+2. Increased thrombin formation was observed with melagatran 119–474 nM or dabigatran 68–545 nM. DTIs did not increase thrombin formation in protein C-deficient plasma, suggesting that both protein C and TM are needed for the DTI-mediated increase in thrombin formation. The results suggest that low concentrations of DTIs suppress the anticoagulant effects of the thrombin–TM/APC system by inhibiting activation of protein C by the thrombin–TM complex, and thereby enhance thrombin formation. Conversely, rivaroxaban does not increase thrombin formation, suggesting that it does not suppress the negative-feedback reaction by inhibition of protein C activation. This hypothesis is supported by the absence of enhanced thrombin formation in protein C-deficient plasma. Enhanced thrombin formation might explain the hypercoagulation observed with DTIs in a rat model of TF-induced intravascular coagulation (Furugohri, et al. 2005; Morishima, et al. 2005; Perzborn, et al. 2008) and suggests that DTIs could cause activation of coagulation at lower plasma concentrations. Table. Effect of rivaroxaban, dabigatran, and melagatran on peak thrombin formation (Cmax [nM thrombin]) in the absence or presence of thrombomodulin (TM) in normal plasma (NP) from healthy volunteers (n=8–12), and in pooled protein C-deficient plasma (PPC) in the presence of TM (n=3). Results were obtained by the CAT method and are a mean of n plasma samples. Prothrombin fragments F1+2 (nM F1+2) results were obtained in the presence of TM (mean results; n=3 [NP]). Thrombin (nM) in normal and in protein C-deficient plasma n.d:, no data. Rivaroxaban (nM) 0 18 91 182 363 1,090 Cmax: − TM in NP 273 222 113 71 43 15 Cmax: + TM in NP 100 67 32 18 10 3 Cmax: + TM in PPC 290 249 168 117 80 38 F1+2: + TM (in NP) 184 89 43 18 n.d. 2 Dabigatran (nM) 0 68 136 273 545 1,090 Cmax: − TM in NP 261 287 289 282 239 102 Cmax: + TM in NP 81 156 203 240 218 96 Cmax: + TM in PPC 298 301 300 292 253 116 F1+2: + TM in NP 252 n.d. 362 422 351 98 Melagatran(nM) 0 24 119 237 474 948 Cmax: − TM in NP 276 290 297 289 251 127 Cmax: + TM in NP 101 133 215 251 237 113 Cmax: + TM in PPC 294 296 299 292 256 132 F1+2: + TM in NP 213 n.d. 389 427 393 123

Antibodies to β2-Glycoprotein I Inhibit Phospholipid Dependent Coagulation Reactions

1993 ◽  
Vol 70 (04) ◽  
pp. 598-602 ◽  
Author(s):  
John T Brandt
Keyword(s):  
Lupus Anticoagulant ◽  
Protein Complexes ◽  
Recent Observation ◽  
Anticoagulant Activity ◽  
Specific Interaction ◽  
Clinical Manifestations ◽  
Dependent Manner ◽  
Prothrombinase Complex ◽  
Glycoprotein I

SummaryLupus anticoagulants are antibodies that inhibit phospholipid dependent coagulation reactions in vitro. These antibodies are of clinical interest because of their association with a variety of clinical manifestations characterized by microvascular thrombosis. Although these antibodies were originally thought to be directed at negatively charged phospholipid, recent studies have suggested that they may be directed at phospholipid-protein complexes. The effect of antibodies directed against β2-glycoprotein I (β2-GP I, apolipoprotein H) on phospholipid-dependent coagulation reactions has been studied. Polyclonal and monoclonal antibodies to β2-GP I were found to inhibit thrombin generation in a dose dependent manner. Inhibition of thrombin formation was due to specific interaction with β2-GP I. There was no evidence that inhibition was due to crossreactivity with other proteins involved in the prothrombinase complex. These findings document that antibodies directed against β2-GP I can have anticoagulant activity analogous to lupus anticoagulant activity and are consistent with the recent observation of such activity in lupus anticoagulant patient samples.

Protamine sulfate down-regulates thrombin generation by inhibiting factor V activation

Blood ◽  
2009 ◽  
Vol 114 (8) ◽  
pp. 1658-1665 ◽  
Author(s):  
Fionnuala Ni Ainle ◽  
Roger J. S. Preston ◽  
P. Vincent Jenkins ◽  
Hendrik J. Nel ◽  
Jennifer A. Johnson ◽  
...  
Keyword(s):  
Human Plasma ◽  
Thrombin Generation ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Protamine Sulfate ◽  
Factor V ◽  
Down Regulation ◽  
Normal Human ◽  
Dose Dependent

AbstractProtamine sulfate is a positively charged polypeptide widely used to reverse heparin-induced anticoagulation. Paradoxically, prospective randomized trials have shown that protamine administration for heparin neutralization is associated with increased bleeding, particularly after cardiothoracic surgery with cardiopulmonary bypass. The molecular mechanism(s) through which protamine mediates this anticoagulant effect has not been defined. In vivo administration of pharmacologic doses of protamine to BALB/c mice significantly reduced plasma thrombin generation and prolonged tail-bleeding time (from 120 to 199 seconds). Similarly, in pooled normal human plasma, protamine caused significant dose-dependent prolongations of both prothrombin time and activated partial thromboplastin time. Protamine also markedly attenuated tissue factor-initiated thrombin generation in human plasma, causing a significant decrease in endogenous thrombin potential (41% ± 7%). As expected, low-dose protamine effectively reversed the anticoagulant activity of unfractionated heparin in plasma. However, elevated protamine concentrations were associated with progressive dose-dependent reduction in thrombin generation. To assess the mechanism by which protamine mediates down-regulation of thrombin generation, the effect of protamine on factor V activation was assessed. Protamine was found to significantly reduce the rate of factor V activation by both thrombin and factor Xa. Protamine mediates its anticoagulant activity in plasma by down-regulation of thrombin generation via a novel mechanism, specifically inhibition of factor V activation.

Effects of Direct Factor Xa Inhibitor, YM150, on Clot Formation and Clot Lysis In Vitro Compared with Other Anticoagulants.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3153-3153 ◽  
Author(s):  
Seiji Kaku ◽  
Ken-ichi Suzuki ◽  
Toshiyuki Funatsu ◽  
Minori Saitoh ◽  
Hiroyuki Koshio ◽  
...  
Keyword(s):  
Human Plasma ◽  
Statistical Significance ◽  
Factor Xa ◽  
Clot Formation ◽  
Thrombin Inhibitor ◽  
Clot Lysis ◽  
Factor Xa Inhibitor ◽  
Dependent Manner ◽  
Direct Factor ◽  
Clotting Time

Abstract The objective of this study was to evaluate the effects of direct factor Xa inhibitor, YM150 and its major in vivo metabolite, YM-222714, on clot formation and clot lysis compared with other anticoagulants, such as a direct thrombin inhibitor (melagatran), a pentasaccharide (fondaparinux), low molecular weight heparin (enoxaparin) and unfractionated heparin. To assess clot lysis, the tissue plasminogen activator (tPA)-induced clot lysis assay was used with human plasma triggered by low and high levels of tissue factor (TF). Under low TF conditions, clot formation was completely prevented by melagatran at 1 μmol/L, by fondaparinux at all concentrations examined (0.1 to 1 μg/mL), by enoxaparin at 0.3 and 1 IU/mL and by heparin at 0.1 and 0.3 U/mL. Even under high TF conditions, 0.3 U/mL heparin prevented any clot formation. Although melagatran, fondaparinux, enoxaparin, and heparin potently prevented plasma clot formation under low TF conditions, under high TF conditions they were less effective at prolonging the clotting time. Under both low and high TF conditions, YM150 and YM-222714 prolonged the clotting time in a concentration dependent manner at concentrations between 0.3 and 3 μmol/L. YM150 and YM-222714 significantly accelerated clot lysis under both low and high TF conditions, but their effects were most evident under high TF conditions. Lower concentrations of melagatran (0.1 and 0.3 μmol/L) enhanced clot lysis under low TF conditions, but under high TF conditions, enhancement of clot lysis required higher melagatran concentrations (0.3 μmol/L or more). Under high TF conditions, fondaparinux enhanced clot lysis only at the highest concentration tested (1 μg/mL). Enoxaparin and heparin enhanced clot lysis under low TF conditions at the lowest test concentrations (0.1 IU/mL and 0.03 U/mL, respectively). Both also enhanced clot lysis under high TF conditions, but their effect reached statistical significance only at higher concentrations (1 IU/mL and 0.1 U/mL, respectively). These results suggested that direct factor Xa inhibitors, YM150 and YM-222714, exert stable anticoagulant effects independently of TF concentration. Both inhibitors enhanced tPA-induced fibrinolysis in human plasma clotted via the extrinsic coagulation pathway. Useful characteristics of YM150 and YM-222714, such as a linear dose response and reliable anticoagulation independent of TF concentration, may lead to the creation of an anticoagulant that is easier to use in the clinical setting than existing products. Potentially beneficial antithrombotic effects, which can be promoted by accelerating endogenous fibrinolytic pathways, may further aid in the prevention or treatment of thrombosis.

Lupus Anticoagulants Form Immune Complexes With Prothrombin and Phospholipid That Can Augment Thrombin Production in Flow

Blood ◽  
1999 ◽  
Vol 94 (10) ◽  
pp. 3421-3431 ◽  
Author(s):  
Susan L. Field ◽  
Philip J. Hogg ◽  
Elise B. Daly ◽  
Yan-Ping Dai ◽  
Barbara Murray ◽  
...  
Keyword(s):  
Calcium Ions ◽  
Flow Reactor ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Plasma Phospholipid ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Lupus Anticoagulants

Lupus anticoagulants (LA) are a family of autoantibodies that are associated with in vitro anticoagulant activity but a strong predisposition to in vivo thrombosis. They are directed against plasma phospholipid binding proteins, including prothrombin. We found that a murine monoclonal antiprothrombin antibody and 7 of 7 LA IgGs tested enhanced binding of prothrombin to 25:75 phosphatidyl serine:phosphatidyl choline vesicles in a concentration-dependent manner. We hypothesized that enhanced binding of prothrombin to phospholipid in the presence of LA IgG might result in increased thrombin production when reactions are performed in flow. Thrombin production by purified prothrombinase components was measured in a phospholipid-coated flow reactor. The flow reactor was incubated with prothrombin, calcium ions, and the IgGs and then perfused with prothrombin, calcium ions, the IgGs, factor Va, and factor Xa. A murine monoclonal antiprothrombin antibody and 4 of 6 LA IgGs from patients with a history of thrombosis increased thrombin production up to 100% over control in the first 15 minutes. In summary, LA IgGs concentrate prothrombin on a phospholipid surface that can augment thrombin production by prothrombinase in flow. These observations suggest that LA might propagate coagulation in flowing blood by facilitating prothrombin interaction with the damaged blood vessel wall.

Procoagulant activity of reversibly acylated human factor Xa

Blood ◽  
1995 ◽  
Vol 86 (11) ◽  
pp. 4153-4157 ◽  
Author(s):  
DL Wolf ◽  
PH Lin ◽  
S Hollenbach ◽  
A Wong ◽  
DR Phillips ◽  
...  
Keyword(s):  
Human Plasma ◽  
Factor Xa ◽  
Procoagulant Activity ◽  
In Vitro Assays ◽  
Dependent Manner ◽  
Clotting Factors ◽  
Clinical Safety ◽  
Active Factor ◽  
Plasma Factor ◽  
Inhibitory Antibodies

The plasma clotting factors used to treat hemophiliacs who have developed inhibitory antibodies have a shared history of limited clinical safety and utility. To improve on existing bypass factors, we have developed a reversibly acylated form of human plasma factor Xa capable of providing a time-dependent release of procoagulant activity. Factor Xa was treated with p-amidinophenyl p′-anisate to generate anisoyl Xa. The chemical modification of the protein involves acylation of the active site serine residue of factor Xa. Anisoyl Xa deacylated in a time, pH, and temperature-dependent manner. Active factor Xa generated on deacylation of anisoyl Xa exhibited amidolytic and prothrombinase complex activities in in vitro assays, the level being comparable to those of untreated factor Xa. When Anisoyl Xa was infused into rabbits, active factor Xa was generated on deacylation of the acylated enzyme, which shortened the activated partial thromboplastin time (APTT) in a dose-dependent manner. The duration of effect on rabbit APTT could be directly correlated to the level of human plasma factor Xa. Because anisoyl Xa bypasses the “tenase” complex that is compromised in hemophilia A and B and is unaffected by inhibitory antibodies, it has the potential to be used as an effective bypass therapy.

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