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It is possible that platelet activation may play a pathogenic role in the increased risk of thrombosis associated with antiphospholipid antibodies (APA). In this study, levels of in vivo platelet activation were measured in 20 patients with primary antiphospholipid syndrome (PAPS) and 30 systemic lupus erythematosus (SLE) patients (14 of whom had secondary APS) using sensitive flow cytometry. Soluble P-selectin levels were also assayed. Platelet CD63 expression was significantly higher in PAPS than normal controls (P = 0·007), as well as SLE patients with and without secondary APS (P = 0·03 and P = 0·002 respectively). PAC-1 binding was significantly higher in PAPS than the control group (P = 0·007) and SLE patients without APS (P = 0·015). Platelet–leucocyte complexes were significantly higher in SLE patients than both PAPS and the control group, and platelet–monocyte complexes were significantly increased in PAPS compared with the control group. (Platelet–leucocyte complexes were also significantly higher than controls in 10 rheumatoid arthritis (RA) patients without APA). Soluble P-selectin levels were significantly higher in PAPS and SLE patients than the control group. Platelet CD62p expression, annexin V binding and platelet microparticle numbers were not increased in PAPS or SLE patients. We conclude that there is evidence of increased platelet activation in PAPS and SLE, and this is important to note as it may have potential therapeutic implications with respect to use of antiplatelet agents in these patients.
The antiphospholipid syndrome (APS) is characterized by the occurrence of thrombosis (arterial or venous), recurrent unexplained fetal loss and/or thrombocytopenia in a patient in whom laboratory tests for antiphospholipid antibody (APA) are positive (Harris, 1987). The presence of APS in patients with no other evidence of auto-immune disease is known as primary APS (PAPS) (Asherson et al, 1989), whereas APS concomitant with other disorders, such as systemic lupus erythematosus (SLE), is called secondary APS. Thrombosis is one of the most common clinical events associated with APA. However, the precise mechanism by which APA may promote thrombosis is unresolved. Many studies highlighting abnormalities of several ‘haemostatic’ factors in APS have been published, and a number of plausible hypotheses reviewed (Triplett, 1993; Santoro, 1994; Esmon et al, 1997; Petri, 1997). One model draws parallels with the thrombosis of heparin-induced thrombocytopenia (HIT) (Arnout, 1996), suggesting that a possible pathogenic interaction between APA and platelets may exist.
Numerous studies investigating platelet activation in APS have been performed, and earlier studies assessed platelet activation by measuring platelet release products such as β-thromboglobulin. The introduction of whole blood flow cytometry has been a major advance as it circumvents many of the problems associated with older methods (Shattil et al, 1987). Platelets can be distinguished easily from other circulating blood cells by the use of platelet-specific antibodies to membrane glycoproteins (GP) such as GPIb or GPIIb–IIIa. Platelet degranulation results in expression on the platelet surface membrane of CD62p (P-selectin) after α-granule release (Stenber et al, 1985; Berman et al, 1986), and CD63, which follows lysosomal and dense granule secretion (Nieuwenhuis et al, 1987; Nishibori et al, 1993). A soluble form of P-selectin can be measured using an immunoassay (Dunlop et al, 1992). The GPIIb–IIIa complex undergoes a conformational change when the platelet is activated, and this can be detected using the IgM monoclonal antibody PAC-1 (Shattil et al, 1985). Platelet-derived ‘microparticles’, which can form as a result of platelet activation, from shear forces or by in vitro freeze–thawing (Owens, 1994), can be enumerated using flow cytometric techniques. Cellular activation, which is accompanied by an increase in intracellular calcium, induces a fast transbilayer movement of all phospholipids known as ‘flip-flop’ (Bevers et al, 1983), which results in phosphatidylserine (PS) exposure on the outer leaflet of the cell. PS can be detected by flow cytometry using the GP annexin V (conjugated to fluorescein). Finally, it is known that thrombin-stimulated platelets bind to leucocytes (particularly monocytes rather than neutrophils (Rinder et al, 1991a,b), hence analysis of platelet–leucocyte complexes provides an additional means of assessing platelet activation. The co-localization of platelets and leucocytes is important in pathophysiological events including inflammation and blood coagulation, and it is well documented that platelet–monocyte interactions can accelerate generation of tissue factor by activated monocytes (Silverstein & Nachman, 1987).
Studies of in vivo platelet activation in APS patients have described increased urinary excretion of thromboxane metabolites (Martinuzzo et al, 1993; Forastiero et al, 1998), accelerated spontaneous platelet aggregation (Wiener et al, 1991), significantly higher levels of platelet CD62p (Fanelli et al, 1997), and a significant increase in the percentage of platelet microparticles (Galli et al, 1993). Previously, we reported a significant increase in platelet CD63 expression in 20 PAPS patients compared with a group of healthy controls (Joseph et al, 1998). In the present study we examined platelet activation in patients with PAPS and SLE using several methods because it is recognized that platelet activation is a complex process and measuring degranulation markers alone may limit the ability to detect platelet activation under all circumstances. This is important to measure as antiplatelet agents form a significant part of the therapeutic regime in APS patients.
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Platelet activation may play an important role in the thrombosis associated with APS. A recent study found increased CD62p (but not CD63) expression in the platelet-rich plasma of PAPS patients when compared with normal plasma (Fanelli et al, 1997); whereas, previously, we have reported a significant increase in median platelet CD63 expression and plasma soluble P-selectin in 20 PAPS patients (Joseph et al, 1998). Because platelet activation is a complex process, it is likely that measuring degranulation markers alone may limit the ability to detect platelet activation under all circumstances. Therefore, in the current study, several platelet activation-dependent changes were measured in order to study a group of PAPS patients as well as SLE patients with and without secondary APS.
In this investigation, there was no significant difference in median CD62p expression among the three groups studied. However, median CD63 expression was significantly higher in PAPS patients compared with the control group and SLE patient group. There are several possible explanations for these results. First, it has been shown that circulating degranulated platelets rapidly lose their surface P-selectin to the plasma pool (Michelson et al, 1996). Therefore, platelets may circulate in an increased state of activation but express normal levels of CD62p. Second, it is known that platelets expressing higher levels of CD62p bind preferentially to leucocytes (monocytes and neutrophils) (McEver, 1991), and so would have been excluded from this flow cytometric analysis. However, we were unable to conclude that this preferential binding to leucocytes necessarily results in a reduced number of CD62p-positive single platelets. Median CD63 expression was significantly higher in PAPS patients than in SLE patients with secondary APS. One explanation for this result is that the majority of SLE patients were receiving immunosuppressive therapy at the time of the study, which could potentially ameliorate a platelet-activating/degranulating action of APA.
PAC-1 binding was significantly increased in PAPS, although one SLE patient with secondary APS had the highest level measured. In comparison with results for CD63 expression, there was no significant difference in levels of PAC-1 binding between PAPS and SLE patients with secondary APS. This may be because PAC-1 binding is a more ‘sensitive’ index of platelet activation than the expression of degranulation markers (Bihour et al, 1995).
There was no significant difference in median levels of annexin V binding between the control group, PAPS and SLE patients, although some individuals had increased values. This is not unexpected as it is unlikely that significant aminophospholipid exposure occurs under normal resting conditions. In vitro experiments demonstrate that strong agonists such as calcium ionophore A23187 and collagen and thrombin increase the number of annexin V binding sites on platelets (Thiagarajan & Tait, 1990). It is also possible that highly ‘reactive’ PS-expressing platelets are rapidly cleared from the circulation, or that APA may interfere with the binding of annexin V to PS (Rand et al, 1998).
Microparticle numbers were not significantly increased in PAPS or SLE patients, although several patients had elevated levels. Similar to the findings for annexin V binding, it may be that microparticle numbers are not increased in resting conditions, as there is only extensive in vitro formation of platelet microparticles after exposure to calcium ionophore A23187, collagen plus thrombin or complement C5b-9 (Zwaal et al, 1992). Alternatively, microparticles may be cleared rapidly from the circulation. There have been very few reports of microparticle numbers in APS – one group reported a significant increase in mean percentage of microparticles in 11 APA patients (10%) compared with a normal control group (5·5%) (Galli et al, 1993).
Platelet–granulocyte and platelet–lymphocyte complexes were significantly higher in SLE patients compared with PAPS patients and the control group, and platelet–monocyte complexes were significantly elevated in both patient groups. Platelet–leucocyte complexes enable leucocytes to be involved in haemostasis and thrombosis, and platelet–monocyte interactions may accelerate generation of tissue factor by activated monocytes (Silverstein & Nachman, 1987). Previously, one group reported increased platelet–leucocyte aggregates in SLE and APS patients (Specker et al, 1998). Although it is known that activated platelets bind to leucocytes, it is unlikely that this is the only mechanism for their formation. Evidence of platelet activation was found more often in PAPS than SLE patients, yet, generally, SLE patients had higher percentages of circulating platelet–leucocyte complexes. It may also be that platelet–leucocyte complexes are a feature of auto-immune disease (as part of an inflammatory response), or that they are not cleared from the circulation as rapidly because the reticuloendothelial system may be ‘blocked’ as a result of immune complex deposition. For this reason, patients with RA were also studied. Only platelet–leucocyte complexes were analysed in these patients, hence one cannot exclude the presence of platelet degranulation. However, it is unlikely to have been any more significant than that found in SLE patients without secondary APS. The median values of platelet–granulocyte and platelet–monocyte complexes were significantly higher in RA patients than the control group, suggesting that platelet–leucocyte complexes may be increased in auto-immune–inflammatory diseases in general. The role of leucocyte stimulation in the formation of platelet–leucocyte complexes is somewhat unclear. One group have found that selective activation of leucocytes by N-formyl–methionyl–leucyl–phenylalanine (fMLP) resulted in no increase in the percentage of activated leucocyte-resting platelet conjugates (Rinder et al, 1994), whereas another group demonstrated that fMLP alone increases platelet–leucocyte aggregates dose-dependently in unfixed whole blood (Li et al, 1997). In general, there appeared to be a correlation between the platelet count and level of platelet–leucocyte complexes, as well as between the different subtypes of platelet–leucocyte complexes. These variables may be interdependent in a way not fully described by correlations, therefore we cannot make major inferences. However, it is unlikely that an increased platelet count alone is responsible for the increased platelet–leucocyte complexes seen in the patient groups, as there was no significant difference in platelet counts among the control and patient groups.
Plasma levels of soluble P-selectin were significantly elevated in both PAPS and SLE patients compared with the control group, and were significantly higher in PAPS than in SLE patients. Because soluble P-selectin is a marker of platelet (and possibly endothelial cell) activation, this is consistent with the previous findings of increased platelet activation primarily in PAPS.
In summary, the observations made in this study demonstrate an elevated level of platelet activation in PAPS, as seen by increased expression of platelet CD63, PAC-1 binding and platelet–leucocyte complexes. The finding that levels of other platelet activation markers (i.e. annexin V binding and numbers of microparticles) were not significantly elevated, indicates that the type of platelet activation in PAPS is limited, and does not appear to involve increased levels of platelet aminophospholipid exposure (although this cannot be completely excluded). Although we cannot state for certain, it is unlikely that any of these observations occurred as a result of thrombosis because all patients were stable when studied and had not suffered an acute thrombotic event for months or years. Aspirin did not appear to have a significant effect on expression of the platelet activation markers studied, but patient numbers were small. This may be of clinical significance, however, as it may signify that aspirin alone may not necessarily be the ideal antiplatelet drug of choice in APS. Despite having a lesser degree of increased platelet activation, SLE patients had the highest levels of circulating platelet–leucocyte complexes, and this may be related to factors such as leucocyte activation as well as reduced clearance of these complexes from the circulation. An increase in platelet–leucocyte complexes was also found in RA patients without APA. Whether or not the observed increase in platelet activation in this study is caused by APA IgG is still unclear, and remains the topic of further study.