Procoagulant Platelet Derived Microparticles

Platelet activation by various agonists leads to procoagulant alterations of the platelet membrane. This includes loss of the usual membrane asymmetry (i.e., with platelet activation, there is increased transbilayer movement of phosphatidyl-serine from the inner to the outer leaflet of the platelet plasma membrane). The membrane "flip-flop" is a consequence of a calcium-dependent enzyme ("scramblase") that serves to undo the membrane asymmetry actively maintained in resting platelets by other enzymes (aminophospholipid translocase and "floppase") (Bevers et al., 1999). Additionally, platelet activation also leads to profound morphological changes that include the generation of procoagulant platelet-derived "microparticles" (Sims et al., 1989).

Serum and purified IgG from patients with HIT, as well as immune complexes and murine platelet-activating monoclonal IgG, also generate platelet-derived microparticles via the platelet FcgRIIa; in contrast, quinine- and quinidine-depen-dent sera do not produce microparticles, even though they lead to far greater drug-dependent binding of IgG to platelets, compared with HIT samples (Warkentin et al., 1994). Indeed, HIT serum is superior in generating platelet-derived micro-particles and in producing platelet procoagulant activity than thrombin, collagen, and adenosine diphosphate (ADP); only the nonphysiological agonist calcium ionophore produces greater numbers of microparticles and procoagulant activity than does HIT sera (Warkentin and Sheppard, 1999).

Flow cytometry using particle size ("forward scatter") and fluorescein-labeled platelet, GP-specific monoclonal antibodies can detect platelet-derived microparticles generated by HIT antibodies (Warkentin et al., 1994; Hughes et al., 2000). This technique has been used as a diagnostic assay for HIT (Lee et al., 1996). Alternatively, Tomer (1997) used fluorescent-labeled annexin V, a protein that binds to phosphatidylserine, to detect activated platelets and microparticles (see Chapter 10).

There is some uncertainty as to whether the "microparticles" detected by flow cytometry represent true microparticles or rather platelets that have undergone considerable morphological changes during activation. Use of orthogonal light scatter, combined with fluorescence gating on platelet antigens, detects significant increases in total particle count, suggesting that at least some micro-particles are generated (Bode and Hickerson, 2000). Moreover, microparticles being generated by HIT antibodies is suggested by a study that used confocal microscopy and scanning/transmission electron microscopy for their detection (Hughes et al., 2000) (Fig. 1).

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