Antiphospholipid antibodies: Paradigm in transition

OBJECTIVES: This is a critical review of anti-phospholipid antibodies (aPL). Most prior reviews focus on the aPL syndrome (APS), a thrombotic condition often marked by neurological disturbance. We bring to attention recent evidence that aPL may be equally relevant to non-thrombotic autoimmune conditions, notably, multiple sclerosis and ITP. ORGANIZATION: After a brief history, the recent proliferation of aPL target antigens is reviewed. The implication is that many more exist. Theories of aPL in thrombosis are then reviewed, concluding that all have merit but that aPL may have more diverse pathological consequences than now recognized. Next, conflicting results are explained by methodological differences. The lupus anticoagulant (LA) is then discussed. LA is the best predictor of thrombosis, but why this is true is not settled. Finally, aPL in non-thrombotic disorders is reviewed. CONCLUSION: The current paradigm of aPL holds that they are important in thrombosis, but they may have much wider clinical significance, possibly of special interest in neurology

Clinical and neuroimaging correlates of antiphospholipid antibodies in multiple sclerosis: a preliminary study

<p>Abstract</p> <p>Background</p> <p>The presence of antiphospholipid antibodies (APLA) in multiple sclerosis (MS) patients has been reported frequently but no clear relationship between APLA and the clinical and neuroimaging features of MS have heretofore been shown. We assessed the clinical and neuroimaging features of MS patients with plasma APLA.</p> <p>Methods</p> <p>A consecutive cohort of 24 subjects with relapsing-remitting (RR) MS were studied of whom 7 were in remission (Rem) and 17 in exacerbation (Exc). All subjects were examined and underwent MRI of brain. Patients' plasma was tested by standard ELISA for the presence of both IgM and IgG antibodies using a panel of 6 targets: cardiolipin (CL), β2 glycoprotein I (β2GPI), Factor VII/VIIa (FVIIa), phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylethanolamine (PE).</p> <p>Results</p> <p>In exacerbation up to 80% of MS subjects had elevated titers of IgM antibodies directed against the above antigens. However, in remission, less than half of MS patients had elevated titers of IgM antibodies against one or more of the above antigens. This difference was significant, p < 0.01, for all 6 target antigens. Interestingly, none of the MS patients had elevated plasma titers of IgG against any of the target antigens tested. Correlation analysis between MRI enhancing lesions and plasma levels of APLA revealed high correlation for aPC, aPS and aFVIIa (p ≤ 0.0065), a trend for aPE and aCL (p = 0.056), and no correlation for aβ2GP1. The strongest correlation was for aFVIIa, p = 0.0002.</p> <p>Conclusion</p> <p>The findings of this preliminary study show that increased APLA IgM is associated with exacerbations of MS. Currently, the significance of this association in pathogenesis of MS remains unknown. However, systematic longitudinal studies to measure APLA in larger cohorts of patients with relapsing-remitting MS, particularly before and after treatment with immunomodulatory agents, are needed to confirm these preliminary findings.</p

Surface Adhesion and Growth of Microaggregates under Flow Condition (II): Clinical Findings in Patients with Thrombosis and Thrombocytopenia

Abstract BACKGROUND. Ideally, thrombophilic states should be evaluated under in vitro conditions as close as possible to in vivo. We have been evaluating cell adhesion to surface and subsequent growth of microaggregates employing a new instrument intended to approach that ideal. We utilized the Diamed Impact-R, in which whole blood is added to a cone-and-plate analyzer (CPA) equipped with an image analyzer, and the resulting microaggregates adhering to the plastic plate after shearing are measured by three parameters: number of object adhering, average size of aggregate, and percent surface coverage (SC) by aggregates. (A companion abstract reports in vitro findings indetifying variables affecting results.) METHODS: Blood was drawn in citrate Vacutainers and 130 uL was applied to the instrument ≤3hr after drawing. The CPA was operated at shear rate 1800 sec−1 with 2min run time. Subjects were also assayed for cell-derived microparticles (MP) from leukocytes (LMP, CD45+), platelets (PMP, CD41+), red cells (RMP, glycophorin+) and endothelium (EMP, CD62E+ or CD31+/CD41−) by flow cytometry. MP which bound FITC-annexin V (AnV) were also measured. We investigated normal healthy controls and 2 groups of patients. Subjects consisted of n=33 normal controls (NC); n=38 with recent thrombosis (TBS) but stable for at least 4 wk, of various subgroups (DVT, APS, TIA, MI); n=35 patients with thrombocytopenia (TP, mostly ITP). RESULTS: In NC, the means ±SD for SC, size, and number of aggregates were 10.3±2.5%, 41.4±14.1 um2, and 1540±381, respectively. In patients with TBS, SC and size were significantly higher (SC: 13.0±3.1%, p=0.0002; size: 54.5±22.9 um2, p=0.006) compared to NC. Interestingly, however, there was no difference in number of aggregates between these groups. In patients with TP, SC and number were significantly lower (SC, 6.3±4.0%, p=0.0001; number, 793±417, p=0.00001) than NC, but there was no difference in size of aggregates. When TBS vs. TP groups were compared, all 3 parameters were higher in TBS (SC, p=0.000001; size, p=0.02; number, p=0.000001). To gauge if the method might be useful clinically, we determined how many patients departed from the NC mean by ≥2SD: best was parameter SC, for which 11/38 (29%) TBS were >2 SD above the mean. On the other hand, 18/35 (51%) TP were >2SD below the mean. We attribute this moderate degree of sensitivity to the large SD (high variability) of the NC group. Lastly, we found that AnV+ MP correlated strongly with parameter SC (R=0.65, p<0.0001), as did EMPCD31+ (R=0.60, p<0.001). The other MP measured did not correlate convincingly with SC, size or number. CONCLUSIONS: Our studies show that in thrombotic patients, parameters SC and size of aggregates but not number of objects were significantly elevated compared to NC. The underlying mechanism of this effect is currently unknown. Our data suggest that cell-derived microparticles, specifically AnV+ MP or EMP, may be involved with increased surface coverage and aggregate size. On the other hand, in TP patients, we observed a lower surface coverage and number of objects. This appears to be due to lower platelet counts. These tests appear promising for evaluating thrombophilic or thrombocytopenic states. However it is required to reduce the high variability by evaluating variables such as shear rate, run time, surface coating, platelet and MP count

Recurrent Thrombosis Is Frequently Associated with Increased Microparticle-Mediated Thrombin Generation

Abstract INTRODUCTION. Microparticles (MP) derived from the plasma membranes of platelets (PMP), leukocytes (LMP), red cells (RMP) and endothelial cells (EMP) are known to be associated with a variety of thrombotic and inflammatory disorders. Most studies have measured their numbers in circulation but few have addressed their function and contribution to clinical thrombosis (TBS). We developed an assay to measure microparticle-mediated thrombin generation (MMTG) and evaluated it in patients (pts) with TBS. We report here that MMTG can distinguish pts at high risk of recurrent TBS from those with stable diseases. METHODS. (a) MMTG assay. Platelet-poor plasma (PPP) was obtained from citrated blood by centrifuging platelet-rich plasma (PRP) at 3000xg for 10min, and total MP were sedimented from 1.0 mL of PPP at 20,000xg for 15min. The supernatant was considered particle-free plasma (PFP). The MP were resuspended in normal pooled PFP containing 25 ug/mL corn trypsin inhibitor, then MP sample was tested for MMTG initiated by calcium employing the calibrated automated thrombogram (CAT) method of Hemker et al in a Fluoroskan Ascent plate reader. Measures recorded were the lag times, peaks, and rates (slope) of thrombin generation based on CAT data. In addition, populations and cell origins of the MP were determined by flow cytometry using cell-specific fluorescent antibodies. (b) Clinical study. Sixty-six pts with TBS were studied: 29 with antiphospholipid syndrome, 11 high homocysteine, 17 hypercoagulable state due to defective clotting factors (FVLeiden etc) or natural anticoagulants (ATIII etc), and 9 idiopathic. Of the total, 25 suffered multiple (2–8) thrombotic events within the 5-yr study period (recurrent TBS); the other 41 had only a single episode (stable TBS). All measures were done at least 4 wk after acute events. There were 34 normal controls (NC). RESULTS. (1) MMTG data: Recurrent TBS group showed the highest MP-dependent thrombin generation (lag = 9.1 min, peak = 299.9 nM, and rate = 120.2 nM/min) followed by stable TBS (lag = 12.4, peak = 244.3, and rate = 84.6), and NC (lag = 13.4, peak = 198.6, and rate = 43.1). The difference between groups (recurrent vs. stable; stable vs. NC; recurrent vs. NC) on all three measures were statistically significant, with exception of lag time between stable and NC. (2) MP profiles: Our data showed that recurrent TBS also had highest counts of EMP, PMP, LMP, and RMP, followed by stable TBS, and NC. This trend parallels MMTG data. (3) Sensitivity of MMTG assay: We found that MMTG rate in recurrent TBS was about triple the NC (120.2 vs. 43.1, p2 SD above normal mean. (4) We analyzed correlations between MMTG measures and MP counts in all TBS patients: the best correlation was LMP with lag time (p=0.00014), or with rate (p=0.008), suggesting that LMP may carry tissue factor to initiate coagulation. PMP also correlated with rate and peak (p=0.01). CONCLUSIONS. To our knowledge, this is the first laboratory assay to differentiate stable from recurrent thrombotic pts. MMTG assay can be utilized as a global screening test for risk of recurrent TBS. Microparticle mediated thrombin generation (MMTG) may play an important role in the pathogenesis of recurrent TBS

Increased Prevalence of Anti-CD36 and Elevated Endothelial Microparticles in Patients with Recurrent Thrombosis

Abstract BACKGROUND: CD36 is a widely distributed transmembrane glycoprotein, called GpIV on platelets, involved in various physiologic processes. It has been implicated in thrombogenic pathologies such as diabetes, atherosclerosis and anti-phospholipid syndrome (APS). It is also an adhesion molecule capable of interacting with collagens and is considered a platelet collagen receptor. We investigated autoantibodies (Ab) against CD36 in TTP [Schultz et al, BrJH 103:849, 1998]. The present study was undertaken to explore relationships of anti-platelet Ab (aPlt-Ab), antiphospholipid Ab (APLA), and circulating cell-derived microparticles (MP) in patients with thrombosis. METHODS: Forty-five patients with documented thrombosis (TB) referred for hypercoagulable workups were recruited consecutively. Of them, 18 suffered from recurrent thrombosis (rTB) while 27 had a single event (sTB) within the past 5 years. We measured APLA, lupus anticoagulant (LA), antiplatelet antibody (aPlt-Ab) and MP. The APLA were IgG and IgM against cardiolipin (CL) and β2-glycoprotein-I (β2GP1) measured by ELISA. The aPlt-Abs were IgG and IgM reacting to GpIIb/IIIa (CD41b), GpIb/IX (CD42b), and GpIV (CD36), assayed by the PAICA method of Macchi et al [Thromb Haemost 76:1020, 1996]. The following types of MP were measured by flow cytometry: endothelial MP defined by CD31+/CD42− (EMP31) or by CD62E+ (EMP62); platelet MP defined by CD31+/CD42+ (PMP); leukocyte MP defined by CD45+ (LMP); and red blood cell MP by glycophorin+ (RMP). Lupus anticoagulant (LA) was measured in the hospital clinical laboratory and was considered positive if 2 of 3 different test methods were positive. Platelet activation was measured by CD62p expression in flow cytometry. RESULTS: Although both groups had increased prevalence of APLA and LA, there were no significant differences between the sTB and rTB groups. About 70% of patients in both groups were positive for at least one APLA. Of the aPlt-Ab, only aGpIV (aCD36) statistically discriminated between the groups, being more frequent in rTB for both IgG (p<0.02) and IgM (p<0.02). Among the MPs assayed, PMP were elevated in all patients in both groups. EMP62 discriminated best between rTB and sTB (p=0.003). LMP and RMP were also significantly higher in rTB than sTB, p<0.025. When we compared patients CD36+ vs CD36− we found that EMP62 was more frequently elevated in CD36+ patients (7/12, 58%) than in CD36− patients (2/32, 6%), p=0.001. Platelet activation marker CD62p was more often increased in rTB than sTB, p=0.022. CONCLUSION: GpIV (CD36) antibodies are the most prominent risk factor for rTB to emerge from this study. This confirms a previous report [Pelegri et al, Clin Exp Rheum 21:221, 2003]. EMP, LMP, RMP and platelet activation (CD62p) are also increased in the recurrent TB group vs single TB. EMP62 were associated with aCD36. Since CD36 is found also on endothelial cells, this suggests that aCD36 could be responsible for the high EMP62 in the rTB group. The present study demonstrates an association between high EMP62 and aCD36. We suggest that persisting autoantibodies against GpIV activate EC to predispose to recurrent thrombosis

Antibodies Against Platelet Glycoprotein Target Antigens in Antiphospholipid Syndrome (APS)

Abstract Introduction: Antiphospholipid syndrome (APS) is characterized clinically by thrombotic events and the presence of antiphospholipid antibodies (aPLA) and/or lupus anticoagulant (LA). It is frequently associated with thrombocytopenia and anti-platelet antibodies have been implicated by some. However the roles of anti-platelet antibodies in APS have not been elucidated. We previously reported that platelet activation, but not endothelial activation, was associated with thrombosis in aPLA+ patients [Blood, 104:143a, 2004] but the cause of platelet activation was not addressed. In the present study, we investigated the prevalence of anti-platelet antibodies in APS patients, as well as platelet and endothelial activation. Material and Methods: We evaluated 47 patients with primary APS. Anti-platelet antibodies against GP IIb/IIIa (CD41b), GP Ib/IX (CD 42b) and GP IV (CD36) for IgG and IgM class were measured by PAICA assay [Thromb Haemost76:1820, 1996]. We also measured platelet and endothelial activation markers by flow cytometry: CD62P on platelets, CD31+/CD42+ platelet microparticles (PMP), and CD31+/CD42- endothelial microparticles (EMP). Results: Of the 47 patients, 34 (72%) were positive for at least one anti-platelet antibody. Looking first at IgG, 18/34 (53%) were positive for GP IV; 17/34 (50%) for GP IIb/IIIa; and 16/34 (47%) for GP Ib/IX. IgM antibodies were 47% (14/30) for GP Ib/IX, 38%(13/34) for GP IIb/IIIa, and 24% (8/33) for GP IV. Platelet and endothelial markers were significantly more common in the anti-platelet antibodies positive group: 40% vs. 21% for CD62P, 40% vs. 28.5% for EMP, and 23% vs. 5% for PMP, respectively. We found that CD62P associated significantly with IgM anti-GP IIb/IIIa (p< 0.05), and PMP with IgM anti-GP IIb/IIIa (p< 0.05), and IgM anti-GP IV (p< 0.05). Conclusions: Anti-platelet antibodies are common in APS, confirming previous reports. We found that anti-platelet antibodies IgM anti-GP IIb/IIIa, and IgM anti-GP IV were often associated with platelet activation, suggesting that these antibodies may activate platelets to play an important role in the thrombogenesis of APS. These antibodies were also associated with endothelial activation. It remains to be determined which antibodies, APLA and/or anti-platelet antibodies, play a dominant role in the activation of platelet or endothelial cells and contibute most to the pathogenesis of thrombosis in APS

Plasma Cholinesterase Activity in ITP Patients with/without Thrombosis

Abstract Abstract 3997 Poster Board III-933 Introduction In the process of immune mediated destruction, platelets may become activated and release procoagulant microparticles in ITP (Immune Thrombocytopenic Purpura). Although bleeding is common manifestation, some patients with ITP may suffer thrombotic complications, often presenting with TIA like syndromes. Brain MRI revealed findings consistent with ischemic small vessel disease in this subgroup (J Lab Clin Med 119: 334, 1992, Thromb Res 107:337, 2002). Recently acetylcoholinesterase (AChE) of RBC and/or non-specific plasma cholinesterase (ChE) was reported to be associated with the inflammation of blood vessels and may be a marker of some inflammatory states. In the present study we investigated plasma ChE activity in patients with ITP with or without thrombotic complication. Methods (i) Patients. We measured ChE activity in 49 patients with ITP. They were sub-grouped as having thrombosis (TBS), F/M (11/7), and non-thrombosis (Non-TBS), F/M (23/8). The TBS group included 9 with TIA like syndrome, 6 with CAD and 3 with venous thrombosis. (ii) Assay of ChE was essentially by Ellman's method. In our system, milli-absorbance units/min (mA/min) x 0.065 = umols substrate cleaved/min. Values reported here are in units of mA/min per mL plasma. Normal controls (NC, n=14) had a cutoff of 3000 mA/min per mL plasma (=mean +2SD). (iii) Sample handling. Platelet-poor plasma (PPP) was prepared by centrifuging 10 min at 1800 xg, then frozen in aliquots. For assay, it was diluted 1:20 with saline, then 5 uL and 10 uL were used in 96-well microtiter plate. Results (i) We observed higher level of ChE in the TBS group compared to Non-TBS in ITP. The TBS group (+/- SD) had mean value 2859 ±866, while the Non-TBS group had 2267 ±777 (units as above). This difference was significant, p3000 (normal cutoff values) compared to only 2/9 (22%) of those with other TBS >3000. Conclusion (i) The ChE activity of ITP patients with TBS is significantly increased compared to Non-TBS in ITP patients. It has been suggested that Blood ChE/AChE may reflect some inflammatory, prothrombotic states. (ii) ITP patients with TIA exhibited higher ChE activity among TBS subgroups in ITP, suggesting that ChE activity may serve as a useful biomarker in TIA like syndrome associated with ITP. However, further study in a larger number of patients is needed to confirm these preliminary findings. Disclosures: No relevant conflicts of interest to declare

Surface Adhesion and Growth of Microaggregates under Flow Conditions (I): Role of Cell-Derived Microparticles and Leukocytes

Abstract BACKGROUND. Circulating blood cells and endothelial cells (EC) shed small membranous vesicles termed cell-derived microparticles (MP) upon activation or apoptosis. Many MP species carry procoagulant activity and cell adhesion molecules (CAM) which can mediate interactions with platelets, leukocytes and EC. Therefore, it has been proposed that some species of MP play important roles in hemostasis and thrombosis. To gain new insight on possible MP modulation of cell adhesion to surfaces under flow conditions, we evaluated effects of adding selected MP types to whole blood (WB) or to platelet-rich plasma (PRP) under flow conditions at physiological shear rates. METHODS. A cone-and-plate analyzer (CPA) equipped with microscope and imaging analysis software was used to investigate cell adhesion and growth of micro-aggregates on the plate surface under flow (shear) conditions (Diamed Impact-R). Citrated WB or PRP (110 μL) was preincubated with 20 μL of selected MP for 10 min, then subjected to 1800 sec−1 shear rate for 2 min. Endothelial MP (EMP) were obtained from the supernatant of cultured renal microvascular endothelial cells activated by TNF-α (10 nM) for 24 hr. Platelet MP (PMP) were generated by exposure of PRP to ADP (10 μM) for 30 min. Red cell MP (RMP) were prepared by exposure of washed RBC to calcium ionophore (10 μM) for 60 min. All MP were washed twice with PBS and resuspended at 2 × 106 /μL. Images of adherent microaggregates were measured in terms of percent surface coverage (SC), average size, and number of adherent objects. RESULTS. The difference between PRP and WB: There was 3-fold greater SC and 2-fold larger aggregate size in WB (p<0.0001, p<0.002, respectively) compared to PRP, but no difference in number of objects. This suggests that platelets act as initial seeds, since number of objects was similar between WB and PRP; and that other cells present in WB contribute strongly to the growth of the initial seeds into larger aggregates. This was further supported by finding that addition of washed leukocytes (but not RBC) to PRP resulted in substantial increase of size and SC, to values similar to WB. Effect of PMP: Addition of PMP to PRP had no effect on any parameter. However, addition of PMP to WB had a strong effect, increasing both SC and size (p=0.01), but not number of objects. We attribute this effect to recruitment of leukocytes in WB by PMP. Effect of EMP: In contrast to PMP, addition of EMP to PRP caused all parameters to increase markedly (SC, p=0.005; size, p=0.002; number, p=0.008). Based on our previous work, we attribute this effect may be similar to the in the oresence ristocetin. When EMP were added to WB, again all values increased compared to WB alone, but less markedly (SC, p=0.001; size, p=0.02; number, p=0.03). Effect of RMP: Addition of RMP to either WB or PRP had no significant effect. CONCLUSIONS. First, we demonstrate that platelets are the initial seeds of aggregate deposition on the plate of this device, and that subsequent growth in size depends on both platelets and leukocytes. Second, we show that both PMP and EMP are active in promoting aggregate growth, but in different ways: PMP apparently act mainly to recruit leukocytes, and EMP act to promote platelet adhesion and growth of aggregates. The underlying mechanisms of MP species on adhesion and growth of aggregates and the physiologic or pathologic significance of these observations is being further explored

Antiphospholipid antibodies and platelet activation as risk factors for thrombosis in thrombocythaemia

Introduction: Risk factors for thrombosis (TB) in thrombocythaemia (TC) associated with myeloproliferative disorder (MPD) are not well defined. Methods: We measured antiphospholipid antibodies (APLA) in 35 patients with TC associated with MPD. Fourteen had TB and 21 did not. We assayed IgG and IgM APLA by ELISA for 6 antigens: β2GP1, cardiolipin (CL), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE) and FVII/VIIa, together with markers of activation of platelets (CD62P) and endothelium [endothelial microparticles (EMP)]. Results: At least one positive APLA was detected in 66% of TC patients overall. The incidence was significantly higher in the TB subgroup (92.8%) than non-TB (47.6%, p < 0.05). Multiple APLA (positive for more than one antigen) were also more frequent in TB, for both IgG and IgM, for all 6 antigens tested (p < 0.05). However, IgM APLA predominated, being about 2-fold more frequently positive than IgG for all 6 antigens. Platelet CD62P was significantly higher in the TB group (p < 0.05). EMP did not differ between TB and non-TB. The most frequent thrombotic complication was recurring ischemic cerebral vascular accidents (ICVA), leading to progressive cognitive impairment. Venous TB often developed at unusual sites. Recurring and reversible TB were common features in TC. Summary: This study suggests that APLA and platelet activation are risk factors for TB in TC. APLA are prevalent in TC, and IgM APLA predominated over IgG. Activation of platelets but not of endothelium may be consistent with the reversible and recurrent features of TB in TC