Platelet microparticles: a wide-angle perspective1

Critical Reviews in Oncology/Hematology 30 (1999) 111 – 142

Platelet microparticles: a wide-angle perspective  Lawrence L. Horstman, Yeon S. Ahn * Wallace H. Coulter Platelet Laboratory, Department of Medicine, Uni6ersity of Miami, Box R36 -A, 1600 NW 10th A6enue, Miami, FL 33136, USA Accepted 7 September 1998

Contents 1. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Orientation and history . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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2. Assay methodologies . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Quantitation of PMP . . . . . . . . . . . . . . . . . . . . . 2.1.1. Total phosphate. . . . . . . . . . . . . . . . . . . 2.1.2. Radiolabeled mAb . . . . . . . . . . . . . . . . . 2.1.3. Flow cytometry (1): direct detection by forward light scatter (FLS) . . . . . . . . . . . . . . . . . 2.1.4. Flow cytometry (2): detection by mAb markers 2.1.5. Flow cytometry (3): PMP quantitation . . . . . 2.1.6. Other methods . . . . . . . . . . . . . . . . . . . 2.2. Procoagulant or ‘‘functional’’ assays . . . . . . . . . . . . . 2.2.1. PF3 activity . . . . . . . . . . . . . . . . . . . . . 2.2.2. Problem of unit of PF3 activity . . . . . . . . . 2.2.3. Prothrombinase assays . . . . . . . . . . . . . . .

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3. Characterization of PMP . . . . . . . . . . . . . . . . . . . . 3.1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Heterogeneity . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Platelet-specific glycoproteins (GPs) on PMP . . . . . . 3.4. Comment on nomenclature . . . . . . . . . . . . . . . . 3.5. Other substances carried on PMP . . . . . . . . . . . . 3.6. Cytoskeleton features—mechanisms of PMP shedding 3.7. Membrane asymmetry . . . . . . . . . . . . . . . . . . . 3.7.1. Annexin V as a PS probe . . . . . . . . . . . 3.8. Procoagulant versus anticoagulant roles of PMP . . . 3.9. Microparticles (MPs) of other cell lineages . . . . . . . 3.9.1. MP from red cells . . . . . . . . . . . . . . . 3.9.2. MP from leukocytes (WBC), endothelium (see also Section 5.3) . . . . . . . . . . . . . 3.9.3. MP from endothelium . . . . . . . . . . . . . 3.10. Synthetic liposomes as model PMP . . . . . . . . . .

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* Corresponding author. Tel.: +1-305-2436703 (6617); fax: +1-305-2435957.  This article is dedicated to the memory of a dear friend and long-time supporter of the author’s laboratory, Wallace H. Coulter, inventor of the Coulter counter, who recently passed away (1913–1998). 1040-8428/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 1 0 4 0 - 8 4 2 8 ( 9 8 ) 0 0 0 4 4 - 4

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L.L. Horstman, Y.S. Ahn / Critical Re6iews in Oncology/Hematology 30 (1999) 111–142 4. Modes and mechanisms of PMP production . . . . . . . . . . 4.1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. By classical agonists . . . . . . . . . . . . . . . . . . . . . 4.2.1. Platelet activating factor (PAF) . . . . . . . . . 4.2.2. Vasopressin, DDAVP . . . . . . . . . . . . . . 4.2.3. Plasmin. . . . . . . . . . . . . . . . . . . . . . . 4.3. PMP by complement (C) in absence of antibodies. . . . 4.4. PMP by anti-platelet Ab with/without complement (C) . 4.4.1. Platelet activation/PMP generation by Abs independent of C . . . . . . . . . . . . . . . . 4.4.2. Importance of epitope . . . . . . . . . . . . . . 4.4.3. Immune complex (IC) . . . . . . . . . . . . . . 4.4.4. Mechanism of FcR activation. . . . . . . . . . 4.4.5. Platelet agglutination . . . . . . . . . . . . . . . 4.4.6. PMP and GP IIb/IIIa Abs . . . . . . . . . . . 4.5. PMP in blood-bank products; platelet storage lesion . . 4.6. PMP arising by shearing stress, foreign surfaces . . . . . 4.7. Voice of dissent. . . . . . . . . . . . . . . . . . . . . . . .

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5. Clinical relevance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Deficient vesiculation: Scott syndrome and related defects . . . . . . . . . . 5.1.1. Scott syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1.2. Inverse Scott syndrome . . . . . . . . . . . . . . . . . . . . . . . . 5.1.3. Castaman’s defect. . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Abnormally elevated vesiculation (PMP production) . . . . . . . . . . . . . 5.2.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.2. PMP in immune thrombocytopenic purpura (ITP) (see also Section 4.4) . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.3. PMP in transient ischemic attacks (TIA) . . . . . . . . . . . . . . 5.2.4. PMP in acute coronary syndromes (ACS). . . . . . . . . . . . . . 5.2.5. PMP versus PF3 activity in thrombosis . . . . . . . . . . . . . . . 5.2.6. Antiphospholipid antibody (aPL), lupus anticoagulant (LAC) . . 5.2.6.1. Relevance to PMP . . . . . . . . . . . . . . . . . . . . . 5.2.6.2. Cytoplasmic Abs and PMP . . . . . . . . . . . . . . . . 5.2.6.3. Platelet activation in aPL . . . . . . . . . . . . . . . . . 5.2.6.4. Other antigenic cofactors in aPL . . . . . . . . . . . . . 5.2.7. Additional evidences of PMP in thrombotic disorders . . . . . . . 5.2.7.1. Thrombotic thrombocytopenic purpura (TTP) . . . . . 5.2.7.2. PMP in heparin-induced thrombocytopenia (HIT), other drug-induced thrombocytopenias . . . . . . . . . 5.2.8. PMP in neurologic disorders . . . . . . . . . . . . . . . . . . . . . 5.2.8.1. Multiple sclerosis (MS) and Alzheimer’s disease (AZ) . 5.2.8.2. PMP in cardiopulmonary bypass (CPB) surgery, hemodialysis, etc. . . . . . . . . . . . . . . . . 5.3. PMP in cell–cell interactions (see also Section 3.9) . . . . . . . . . . . . . .

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6. Summary and concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Clinical significance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. PMP as marker of platelet activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Reviewers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Appendix A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Biographies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Background

2. Assay methodologies

1.1. Orientation and history

Three aspects of PMP assays may be identified: (i) enumerating or otherwise quantifying their population or mass, (ii) measuring their associated functional activity, e.g. procoagulant activity, usually as PF3, and (iii) measuring other putatively functional properties (see Section 3.5).

Platelet microparticles (PMPs) are submicroscopic (  B0.5 mm) membrane vesicles released by platelets during activation, and carry at least some antigens characteristic of intact platelets, chiefly GP IIb/IIIa and GP Ib. They are not detected by ordinary platelet counting methods but may be detected by flow cytometry or other methods reviewed below. PMP were early suspected of having clinical relevance because, like activated whole platelets, they express phospholipids (PPL) which are procoagulant, i.e. furnish a PPL surface essential for assembly of the vitamin-K dependent coagulation factors, notably the prothrombinase and tenase complexes, but also the anticoagulant protein C/protein S system, phospholipase A2 (PLA2), and other PPL-dependent activities. However, the extent of the significance of PMP in various clinical settings has been controversial. The purpose of this review is to summarize the present state of investigations relevant to PMP, including the growing list of clinical disorders associated with PMP abnormalities (see Section 5). It was known since the 1940s that platelet-poor plasma (PPP) coagulated more slowly than platelet-rich plasma (PRP) when calcium was added back, implying a role for platelets [1,2]. However, additional highspeed centrifugation of PPP further prolonged clotting, implying also a subcellular factor [1]. Conversely, O’Brian showed in 1955 that adding serum to PPP shortened the clotting time to even less than that of whole PRP [2], and it was soon found that certain crude phospholipids could substitute for this activity, as further reviewed below. Many of these confusing observations were explained by the discovery of PMP by Wolf in 1967 [3]. He clearly demonstrated that platelets following activation or long standing shed numerous tiny membrane fragments, called by him ‘‘platelet dust’’, which had the same procoagulant activity as whole activated platelets. This activity came to be called platelet factor 3 (PF3) [4]. Transmission electron microscopy (TEM) revealed a range of vesicle (PMP) sizes from B0.1 mm and aggregates of them [3]. Warren and Vales demonstrated in 1972 the release of PMP from platelets following adhesion to vessel walls [5]. Other pioneering investigators of PMP are cited later. PMP are now known to be a normal consequence of platelet activation, and the differences seen in coagulation times between PRP, PPP, and PFP (particle-free plasma) are attributable to platelet- and PMP-associated procoagulant (PF3) activity. This topic has been briefly reviewed by Owens [6], who also used the term platelet microparticles.

2.1. Quantitation of PMP 2.1.1. Total phosphate Many workers have measured PMP released to the supernatant by assay of total PPL in terms of inorganic phosphate (Pi), usually by the conversion factor, 1 mmol Pi = 1 mmol PPL= 774 mg; PPL : 70% of total platelet lipids [7] For example, Butikofer et al. [8] quantified RBC microparticles by Pi using the method of Bartlett, as did Sims et al. for PMP [9]. This is among the less ambiguous methods but is tedious and is restricted to work with isolated PMP or washed or gel-filtered platelets in Pi-free buffers since otherwise spurious Pi and other PPL sources are present.

2.1.2. Radiolabeled mAb George et al. detected PMP arising from platelets bearing 125I-labeled monoclonal antibody (mAb) Tab, anti-GP IIb (CD41), ultracentrifuging the supernatant and washing the ‘‘invisible pellet’’ before counting [10]. They assumed 1 molecule mAb per GP IIb, computed the number per platelet, and then assumed the same distribution on PMP to arrive at an estimate of quantity of membrane material released. 2.1.3. Flow cytometry (1): direct detection by forward light scatter (FLS) Flow cytometry is now the most widely used method in PMP studies, owing to its simplicity and the wealth of information it affords about the population under study. For example, Bode et al. detected PMP in a flow cytometer equipped with 600 mW laser, and also with the aid of a flourescent lipophilic dye, DiOC6, calibrating size with latex beads [11]. Early publications from Ahn’s lab on PMP were also based on direct detection by FLS in a Coulter Epics V flow cytometer equipped with a 5W water-cooled laser, secondarily confirming platelet-derived identity of the particles by FITC labeled mAb [12,13]. In normal volunteers and patients with thrombotic disorders, most microparticles (\ 90%) were platelet-derived, as established by finding \90% positivity for platelet-specific markers. FITC labeled anti-glycophorin revealed few particles of RBC origin (unpublished); see Section 3.9.

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2.1.4. Flow cytometry (2): detection by mAb markers Many clinical flow cytometers now utilize smaller lasers, e.g. the Coulter Profile II has maximum 25 mW laser, and the Coulter XL has a fixed 15 mW laser. Although adequate for most cell studies, these fail to detect all but the largest PMP directly by FLS. For example, when Ahn’s group switched from the older Coulter Epics V to the Profile II, and then to the XL, only about 20% as many PMP could be detected by FLS. Therefore, in subsequent work they turned to primary detection of PMP by the flourescent (FL) signal of an FITC-conjugated mAb label, as described in [14], giving a PMP counting efficiency similar to that by FLS with the big laser (flourescence is detected by a photomultiplier tube, which is more sensitive than the diode used to detect FLS). Unfortunately many authors fail to specify whether primary detection is by light scatter or by fluorescent trigger. Many or most investigators of PMP have employed similar techniques. Wiedmer et al. used biotinylated mAb W5 against GP Ib followed by avidin-PE, and triggered events counted by the red PE signal [15]. We continue to use the method based on FITC anti-GP IIb/IIIa (activation-independent) [14] because of the brighter signal as compared to the less abundant GP Ib. However, these methods may sometimes be misleading due to possible variability in surface density of the antigen in different populations of PMP. For example, Bode et al. discussed variable loss of GP Ib of platelets and PMP [11] (and see their refs. 19 – 23). We have observed interesting but as yet anecdotal differences in apparent PMP in certain patient groups as measured by markers of CD41 vs. CD42. Further refinemenet of methods may include the use of multicolor markers of other antigens common on PMP (see Section 3.5). We have experimented with fluorescent membrane dyes such as Nile Red in efforts to better quantify total PMP [16]. It is our experience that a flow cytometer equipped with a high-power laser is the instrument of choice. 2.1.5. Flow cytometry (3): PMP quantitation One aspect of this topic is relating the number measured in flow cytometry to concentration in the blood. If the machine delivers the sample by screw-driven syringe at known rate this may be calculated. Alternatively the sample may be mixed with a known concentration of RBC or commercially available beads, as in [17 – 19]. Abrams et al. expressed PMP as a ratio or percentage relative to total platelet-specific events [20], as followed by Galli et al. [21], or with modifications in [22 – 24]. These methods have the claimed advantage of tying PMP counts to platelet counts but suffer from ambiguities as can arise by formation of platelet clumps or apparent loss of platelets following stimulation, which facts are obscured in ratios or percentages, as are relevent numbers in thrombocytopenias. Reverter et al.

express results both ways in their Table VII [18]. Probably the major issue is lab-to-lab variation in detection efficiency, which depends on laser power, definition of PMP, fluorescent marker used, centrifuge speed, fixation method, event triggering, etc. This problem could be addressed by a commercial standard such as PMP from sonically disrupted platelets.

2.1.6. Other methods In 1976, Khan et al. measured PMP in a Coulter P64 Channel Analyzer [25]. Solberg et al. measured PMP on a Coulter Model D blood cell counter equipped with small orifices and found a linear relation between PMP and associated PF3 activity (measured by the shortening of coagulation time of normal PPP caused by addition of PMP, see Section 2.2) [26]. Although counting PMP by the Coulter principle (impedence) would be useful, suitable instruments are no longer available, and this method cannot distinguish PMP from vesicles from other sources. Transmission EM (TEM) is not reliable for quantifying PMP but has been useful in revealing morphologies. An ELISA method was recently described [27], discussed later (see Section 6). 2.2. Procoagulant or ‘‘functional’’ assays 2.2.1. PF3 acti6ity The classical PF3 assay was based on Russell’s viper venom (RVV), which contains serine proteases that activate factors V and X. Exposure of PPP to RVV in the presence of Ca2 + causes coagulation, but only in the presence of certain PPLs (see Section 3.7), normally supplied by activated platelets and/or by PMP. Therefore, the rate of coagulation (RVV time, RVVT) in PPP is limited by the availability of suitable PPL, such as supplied by PMP. The semi-purified factor X-activating component of RVV had the trade name Styp6en and the ‘‘Stypven time’’ was widely used to assay PF3 activity [4,28]. Stypven also activates protein C (PC), but much more slowly than the specific PC-activator in the venom of the southern copperhead, Agkistrodon c. contortrix [29,30]. In the early literature it was popular to measure the ‘‘total PF3’’ activity of platelets by measuring the RVVT or Stypven time before and/or after activation by exposure to kaolin or collagen [31,32]. In a typical variant, Polasek activated PRP with kaolin, then after a time stopped the reaction with citrate, removed the clot and measured residual prothrombin by adding thromboplastin [33]. 2.2.2. Problem of unit of PF3 acti6ity Sandberg [34] among others has commented on the difficulty of defining a unit of PF3 activity. Joist [35] cites Spaet [32] as his method of PF3 assay, but included a PF3 standard consisting of sonically disrupted platelets: a plot of log of concentration of platelet

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fragments against log of coagulation time yielded a straight line, as we also have obtained with crude phospholipid [36]. Accordingly, we suggested the following definition of a unit of activity: log[PPL]=5.493− (log t/0.274)

log(units/ml) in cuvette [36]. This definition allows quantitative comparison of PF3 activities and an additive accounting of activities contributed by various plasma components, as was done in [36], finding that about 75% of PF3 activity is attributable to PMP vs. 25% to the platelets, under the specified conditions. Similar experiments by Howard et al. [37] were reported in terms of clotting times in seconds, which, although interesting, preclude quantitative comparison of the activities contributed by the various fractions (PRP, PPP, PFP). An international standard unit would require use of standard PPL (chemically defined) along with available plasma standards, by which the RVV concentration could be adjusted to yield a specified clotting time at a specified concentration of the PPL standard.

2.2.3. Prothrombinase assays Wolf observed that when the PF3 activity of chylomicrons was compared with that of PMP, the former were inactive in the thrombin generation test [38] but were active in the Stypven time, leading him to suggest that the Stypven time may be misleading as an assay of PF3 activity [3]. Relatedly, the fact that certain crude PPL preparations (usually sphingosides from rabbit brain, ‘‘cephalin’’, but other sources have been advocated [39]) can substitute for platelet-derived material in promoting coagulation with Stypven or whole RVV imply that this assay measures only simple PPL dependency, not additional coagulant properties that PMP may possess. For example, Howard et al. showed that PMP have coagulant effects additional to those of PPL alone, including significant influence on the activated partial thromboplastin time (APTT) and dilute simplastin time test (DSTT) [37]. Similarly, Bode and Miller pointed out that ‘‘the Stypven PF3 assay used by Champion [their ref. 12] to study stored platelets is known to respond non-specifically to neutral phospholipids and lipoproteins [their ref. 13]… cannot be relied upon for comparison of PF3 levels between different concentrates’’ [40]. For these reasons they employed the chromogenic thrombin substrate, S-2238, developed by Sandberg, with purified components of RVV to measure the PPLdependent production of thrombin [40]. Their standard PF3 was the supernatant of freeze-thawed platelets. The principle is well explained in Sandberg et al. [34]: when factor X is activated by Stypven, the rate-limiting factor in thrombin generation is PF3. They remark that PF3

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has never been unambiguously characterized but ‘‘is commonly defined, however, as an activity originating from platelets appearing during the release process and replaceable in clotting tests by phospholipid vesicles of certain compositions’’ [34]. The assay used by Wiedmer et al. [15] also limits the system to known quantities of purified factors, and measures thrombin produced by its action on a chromogenic substrate. They employed mAb V237 to monitor factor Va binding sites on PMP. Variations on such methods abound, notably in employing reversible thrombin inhibitors to prevent feedback inactivation by anti-thrombin; Swords et al. employed DAPA [24], and Oparkiattul et al. used MD805 [41], as explained in [42]. On the other hand, there are trade-offs between efforts at sophistication vs. simplicity, economy, depending on aims. We have obtained useful insights on PF3 activities using whole RVV, as in [36].

3. Characterization of PMP

3.1. Introduction General statements on PMP must be taken with caution because of evidence that they vary in composition, size range, surface antigen expression, and PF3 or other activities, depending on clinical setting or circumstances of their production. The following thumbnail calculations may be helpful for orientation.

3.2. Heterogeneity Assuming that the minimum diameter of a membrane vesicle is twice the thickness of the membrane bilayer, ˚ (=0.02 then small PMP will have diameter 200 A mm), devoid of cytoplasm and practically micelles. As earlier cited, PMP of this size range have been observed in TEM, but are probably invisible to flow cytometry, although high-power lasers might discern them. At the other extreme are what may be called ‘‘mini-platelets’’, arbitrarily discriminated from the normal platelet population in flow cytometry on the basis of size (light scatter) or fluorescent intensity of the label. In the absence of clear definition, the meaning of ‘‘PMP’’ is somewhat arbitrary. If the geometry of the platelet is approximated by a disc of radius r and thickness t= 1/3 diameter d, then if volume V= 6.5 fl (typical MPV), its disc diameter d= 2.9 mm, t= 0.97 mm, and surface area A=22.3 mm2 (the platelet of mean volume is larger than the numberaverage diameter). If this area is entirely converted to spherical PMP of diameter d= 0.02 mm, then about 17 700 PMP result. If the PMP have d= 0.1 mm, about 700 result, yet account for only 6% of the original platelet volume, and its discoid diameter falls only

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slightly, from the original 2.9 – 2.8 mm, a decrement scarcely detectable. This is consistent with many observations showing copious PMP production with little change in platelet population or size, e.g. Bode et al. observed increasing PMP with time of platelet storage but little decline in platelet number or size [40], and we observed much the same for the early stages of complement-mediated lysis [14]. This also implies that the platelet must be capable of supplying additional membrane area from within (granules, open canalicular system, OCS) to compensate for that lost as PMP. It is not always clear whether PMP arise from the complete conversion of a few platelets to PMP or from the partial conversion of many or most of them, but it is likely that either may occur. There is growing evidence that platelets themselves consist of heterogeneous populations, not attributable solely to senescence [43– 48].

3.3. Platelet-specific glycoproteins (GPs) on PMP More than 40 GPs are known to be present on the resting platelet surface [49 – 51], recently including a chemokine receptor [52], and others appear following stimulation, notably CD62 (P-selectin, aka GMP-140 or PADGEM) [53], CD63 (aka GP53) cited in [54], and p-133 (multimerin) [55 – 58]. Many are routinely identified on PMP, chiefly GP IIb/IIIa and GP Ib since they are widely used as markers to identify PMP as such (platelet-derived). George et al. have quantified many GPs on PMP [53], as has Fox et al. [59]. GP IIb/IIIa is modified on activated platelets to a form facilitating fibringogen binding, and certain mAbs (e.g. PAC-1) react preferentially with the activation-dependent epitopes, as referenced in [60]. It was recently shown that PMP can bind fibrinogen and participate in thrombus formation [61].

3.4. Comment on nomenclature Many platelelet membrane GPs are now classified as integrins, consisting of combinations of homologous chains, resulting in a nomenclature by which, for example, GP IIb/IIIa is called aIIbb3 [62 – 66]. However, most publications continue the use of GP IIb/IIIa (sometimes mixed with integrin nomenclature for other platelet GPs); in this review we use the nomenclature traditional in hematology, which stems historically from their patterns in electrophoresis.

3.5. Other substances carried on PMP Despite their diminutive size, platelets contain and can secrete upon activation an astonishingly large number of substances, chiefly from their dense and a-granules [67–70], the most recent addition being VEGF [71].

At least some of these tend to stick on the surface of activated platelets, e.g. thrombospondin, cited in [54], and many are found also on PMP. Fox has partially characterized the vesicles (PMP) resulting from exposure of platelets to calcium ionophore A23187 and found, in addition to a list of surface GPs, many proteins characteristic of the a-granules (thrombospondin, platelet factor 4, b-thromboglobulin, fibrinogen) as well as actin, actin fragments, and talin, all apparently in consequence of calpain hydrolysis [59]. Similar findings were seen when platelet activation was by dibucain ‘‘except that a-granule proteins were not present’’ [59]—another example of heterogeneity, i.e. differing PMP constitution depending on mode of generation. Iwamoto et al. recently found that nearly 90% of the total platelet activating factor (PAF) released from platelets stimulated by thrombin+ collagen was associated with PMP [72]. Nomura et al. identified b amyloid precursor protein (APP) on certain PMP and discovered that APP-positive PMP are particularly thrombogenic [73] (although another report attributes anticoagulant properties to APP [74] and ref. 9 therein). Kelton et al. reported that the Ca2 + -dependent protease calpain is associated with the elevated PMP in active thrombotic thrombocytopenic purpura (TTP) but not when TTP is in remission [75]. Pasquet et al. have more recently reported on calpain in PMP [76], and also refer to PMP as a ‘‘preferential substrate’’ for secretory phospholipse A2 (sPLA2), which generates the lipid mediator lysophosphatidic acid, with many biological effects [77]. Other agents found preferentially associated with PMP are discussed and referenced later, including the anticoagulant proteins C/S (see Section 3.8); fibrin (see Section 4.2.3); complement (C) components (see Section 4.3); the anticoagulant cofactor of anti-PPL Abs, b2GPI (see Section 5.2.6); glycocalicin (see Section 4.5); and probably many others, as suggested by studies of microparticles of other cell lineages, e.g. GPI-anchored proteins like acetylcholinesterase and decay accelerating factor (DAF) on RBC MPs, and thrombomodulin on leukocyte MPs (see Sections 3.9 and 5.3). In addition to these, of course, are the Gladomain containing coagulation factors (vitamin K dependent) which assemble in Ca2 + -dependent fashion on suitable PPL surfaces, notably on activated platelets and PMP. These observations imply that the putative role or function of PMP may go well beyond PF3 activity.

3.6. Cytoskeleton features— mechanisms of PMP shedding In addition to a spectrin-rich membrane skeleton similar to that of RBC, which in platelets is closely associated with GP IIb/IIIa [78], platelets possess a

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network of filaments throughout the cytoplasm, chiefly of actin, readily separated from the membrane skeleton by differential centrifugation following Triton X-100 treatment, and showing distinct changes in composition and structure following platelet activativation or aggregation, as reviewed by Fox [79] and in [80,81]. The presence of contractile proteins in PMP was apparently first demonstrated by Crawford in 1971 [82]. Fox et al. have presented evidence that the platelet membrane skeleton in resting platelets stabilizes against vesiculation and that ‘‘shedding of the microvesicles correlated with the extent of disruption of the membrane skeleton’’, i.e. of actin-membrane interactions; and that when ‘‘disruption… was inhibited by inhibition of calpain, the shedding of the microvesicles was inhibited’’ [59]. Those findings have been extended by Basse et al., who used calpeptin to inhibit calpain-mediated cytoskeleton proteolysis otherwise induced by Ca2 + with A23187, finding that ‘‘vesiculation strongly depended on cytoskeletal proteolysis’’ [83]. They found morphological differences in the filopods of activated platelets in the presence vs. absence of calpeptin following A23187 activation and concluded that the vesicles (PMP) result from fragmentation of the filopods, supporting that hypothesis with SEM. Vesicles liberated were quantitated by measuring phospholipid (PPL) as Pi. More recently, elevated Ca2 + was visually localized to the filopods undergoing vesiculation [84]. Yano et al., too, concluded that PMP are formed by fracture of the budding pseudopods (‘filopods’) upon activation, and noted that cytochalasin D inhibited PMP formation ‘‘almost completely’’ [85]. They also investigated the effect of protein phosphatase inhibitors (calyculin A, okadaic acid) on PMP formation, finding that they caused a doubling of PMP arising from A23187 activation as measured by flow cytometry (550 mW laser), further supporting a key role for cytoskeleton dynamics in PMP formation. Despite all of the above, however, understanding of PMP shedding remains hazy. Additional relevant work is cited later (e.g. see Sections 3.9 and 6).

3.7. Membrane asymmetry Following the lipid bilayer hypothesis of Danielli and Davson [86] it emerged that the inner and outer leaflets of the plasma membrane were different (asymmetric), as first shown in resealed RBC membranes (ghosts) prepared inside-out, resulting in reversed transport of certain pumps, e.g. [87]. Membrane asymmetry was fully appreciated in studies on mitochondria in the 1960s–1970s evaluating Mitchell’s chemiosmotic hypothesis, particularly in studies of ‘‘submitochondrial particles’’, which often were inside-out, e.g. [88,89]. With specific regard to platelets and PMP, the role of membrane sidedness, PPL composition, and electric

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charge has been most vigorously investigated by Zwaal, Comfurius, Schroit, Bevers, and colleagues, as reviewed in 1990 [90] and 1997 [91]. Briefly, the outer leaflet of platelets and ‘‘most if not all eukaryotic cells’’ are rich in the choline PPLs (sphingomyelin, phosphatidylcholine), which do not support coagulation, whereas the inner leaflet is rich in amino PPLs (phosphatidylethanolamine, PE; phosphatidylserine, PS) which are more negative (anionic) at physiologic pH. Zwaal et al. showed that a key event of platelet activation consists of the translocation of normally in-facing anionic PPLs, especially PS, to the outside, where their exposure promotes cell–cell interaction and supports coagulation in the presence of calcium. The PMP shed during activation are also enriched in PS, accounting for their PF3 activity, but exceptions may occur (see Section 3.8). It has long been recognized that anionic PPLs are more active in promoting assembly of the tenase and prothrombinase complex, i.e. in promoting coagulation. Shroit and Zwaal cite in [90] kinetic studies showing that anionic PPLs decrease the Km of factor X and thrombin from far above to far below their plasma concentrations, resulting in more than 106-fold increase in rate of thrombin production [92] and that PS is most potent in promoting coagulation through PF3-like activity [93]. Zwaal has defended the hypothesis of Seigneuret and Devaux [94], that this transbilayer movement or ‘‘flip-flop’’ of PS is accomplished by a specific amino PPL translocase; efforts to purify this and related hypothetical enzymes (scramblase, floppase, [91]) were long elusive but scramblase has recently been sequenced and cloned [95]. It was further proposed that the role of Ca2 + is to dehydrate the microenvironment since Ca2 + but not Mg2 + can eliminate water from the PS microenvironment to promote fusion of storage granules with the plasma membrane, and of the necks of blebs in vesiculation of pseudopodia. References are given in [91] showing that Ca2 + causes PS exposure also in RBC, as commented below (see Section 3.9). It has been shown that collagen induces exposure of anionic PPL in the resulting PMP [96]. In a 1992 review, Zwaal et al. focus specifically on PMP with emphasis on the role of membrane lipids [97]. In a study utilizing doppler electrophoretic light scattering analysis (DELSA), Jy et al. confirmed and extended earlier work showing that normal platelets activated in vitro, as well as platelets from patients with thrombosis and chronic platelet activation, have sharply more negative electrophoretic mobilities than resting controls, with almost diagnostic discrimination [98]. Comfurius et al. showed that ‘‘removal of intracellular calcium produces a decrease of procoagulant activity of the remnant cells but not that of the shed vesicles’’, suggesting to them that the putative amino PPL translocase is absent in the shed PMP but is still

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present and active in the remnant cells, enabling restoration of resting asymmetry [99].

3.7.1. Annexin V as a PS probe Because annexin V (AnV) binds specifically to anionic PPL (in Ca2 + dependent manner), notably to PS, Thiagarajan and Tait exploited AnV labeled with FITC as a probe for exposure of procoagulant PPL and observed that ‘‘collagen-induced exposure of annexin V binding sites correlated directly with increased ability to support activity of the reconstituted prothrombinase complex’’ [96], and that ‘‘staurosporine inhibited collagen-induced but not A23187-induced annexin V binding…’’, leading to their conclusion that ‘‘the procoagulant effects of platelets and platelet-derived microparticles [PMP] is mediated by calcium-induced exposure of anionic phospholipids’’. This use of annexin V is increasing, e.g. [76,100–103] (it has long been used as a marker of apoptoptic or senescent cells). 3.8. Procoagulant 6ersus anticoagulant roles of PMP Contrary to the usually accepted role of PMP in promoting coagulation, it was shown by Tans et al. that some species of PMP may act to inhibit coagulation, by virtue of accelerating the inactivation of factor Va by activated protein C (aPC) [104]. Briefly, they report that the rate of inactivation of Va relative to non-stimulated platelets was 4-fold following ADP or adrenaline (epinephrine); 11-fold following thrombin; 29-fold following collagen; and 60-fold following A23187; and that  25% of this anticoagulant activity was attributable to PMP [104]; see also [19]. Bode and Lust showed a masking of heparin activity by the procogaulant activity of PMP [105]. Other reports were earlier cited showing that the action of PMP in coagulation cannot be attributed solely to PF3-like PPL (see Sections 1 and 2.2). Another means by which the procoagulant activity of PMP may be modulated is suggested in [97] and elsewhere by those authors: not all PMP may display procoagulant PPL, owing to incomplete flip-flop of the membrane asymmetry or to scramblase activity in some PMP species. These observations underscore PMP heterogeneity.

3.9. Microparticles (MPs) of other cell lineages Erythrocytes (RBC, or E), leukocytes (WBC) and endothelial cells are all known to shed MPs under certain circumstances, and may contribute thrombogenic activities similar to PMP. Chylomicrons may also contribute such activities in some clinical settings. Studies with RBC vesicles and synthetic liposomes have been helpful in developing a better understanding of the causes and consequences of circulating MPs.

3.9.1. MP from red cells Erythrocytes (E) have long served as a model for the study of cell membranes, and this has been true also of studies of vesiculation—shedding of E microparticles (EMP)—since the 1970s [106–108]. It was early noted that E suffer a ‘‘storage lesion’’ similar to that of platelets (see Section 4.5): spectrin-free vesicles are released after ]12 h at 37°C in the absence of glucose; but if the level of endogenous ATP is maintained, vesiculation is ‘‘completely inhibited up to 54 h’’ [108]. These vesicles had a mean diameter of 0.185 mm, contained similar amounts of membrane proteins (e.g. glycophorin) as E per unit of membrane area (by total Pi) and had similar PPL content, except for a 10-fold enrichment of phosphatidic acid [108]. Studies by Allan et al. using Ca2 + /A23187 yielded EMP different in certain respects (e.g. spectrin content) from those produced by aging [106]. E ghosts and EMP are known to exhibit PF3-like activity, apparently due to asymmetry-promoting mechanisms similar to those for platelets/PMP, according to Franck et al. [109], where the proposed mechanism is similar to that indicated also by Fox, earlier cited. Despite the abundance of E in circulation, it is our experience that EMP are rare compared to PMP, barring hemolytic disease or abnormal RBC. Fox points out that a variety of hereditary defects involving the membrane skeleton have been implicated in shedding of EMP [59], see also [54]. Procoagulant EMP occur in sickle cell disease [109–111] and in thallasemias, e.g. [112], possibly accounting for, or at least contributing to, the thrombotic complications of those disorders; the latter find that the high PMP-associated PF3 activity is corrected by splenectomy. Toumbis et al. [111] showed in vitro that deoxygenated sickled RBC added to PRP promote release of PF3, assayed by RVV according to Spaet et al. (their ref. 10), suggesting this could promote vasocclusive crises. RBC from b-thalassemia had similar effects [113], i.e. activated platelets; and whole RBC from patients are also procoagulant [114]. Franck et al. performed extensive studies of the prothrombinase activity of the spiculederived EMP released from repeatedly sickled E [109]: E spicule MP are enriched in external PS and PC, presumably accounting for their procoagulant activity. Their experiments included studies of rates of transbilayer exchange of lipids in normal and sickled E, and on inferred effects of a putative PC-specific PPL exchange protein from beef liver. Many studies on the interaction of RBC with defined liposomes have been done, e.g. [115–117], aimed at understanding factors such as dietary lipids which might promote generation of procoagulant EMP. For current work of related interest see [118]. 3.9.2. MP from leukocytes (WBC), endothelium (see also Section 5.3) The intimate interactions between platelets and WBC has been increasingly appreciated in recent years [119].

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These interactions include aspects of hemostasis. Tracy et al. has compared the procoagulant activities of peripheral blood cells [120] and Altieri has more recently reviewed this topic [121]. Stimulated monocytes are known to express tissue factor (TF), e.g. [122], and to shed MP with procoagulant activity [123]. It has been shown that thrombomodulin (TM) is enriched in WBC MPs [124]. Nomura et al. investigated CD68-positive MPs in ITP [17]. Jy et al. gave evidence that PMP preferentially bind to and appear to link PMN into grape-like bunches [125]. Relatedly, Barry et al. reported that PMP may ‘‘activate monocytes and/or endothelial cells’’, and proposed that PMP known to arise in inflammation ‘‘may facilitate adhesive interactions between monocytes and endothelial cells’’ [126].

3.9.3. MP from endothelium Hamilton et al. showed that C5b-9 complex could induce generation of procoagulant MPs from endothelial cells grown in culture, whereas other agonists, including even high concentrations of A23187/Ca2 + , were much weaker in this respect [127]. They concluded that ‘‘the capacity of the C5b-9 proteins to induce vesiculation of the endothelial plasma membrane and thereby expose catalytic surface for the prothrombinase enzyme complex may contribute to fibrin deposition associated with immune endothelial injury’’ [127]. One may conjecture that some so-called soluble receptors may actually be carried on MPs.

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charged membrane surfaces are most efficacious in procoagulant activity, Rosing et al. showed in 1988 that electrostatic charge alone was not the key factor, since by introducing graduated mole fractions of stearylamine to PPL vesicles (often called bangosomes in the older literature, in honor of A.D. Bangham) they were able to produce vesicles bearing net positive charge, which nonetheless were highly efficient in prothrombinase activity [93]. What was crucial, however, was the presence of PS, since as little as 3% mole fraction of PS gave maximal activity, whereas its absence gave zero activity [93]. Loughrey et al. found that the binding of phosphatidylglycerol (PG) liposomes to rat platelets depended on complement, at least C3 [132]. Administration of PG to rats caused a transient thrombocytopenia and formation of platelet microaggregates associated with platelets sequestered in lung, spleen and liver [132,133], reminiscent of platelet consumptive microangiopathies. However, it is not clear if these observations apply to humans since rodent platelets have C3 receptors (CR1, aka CD35) and their RBC do not, the reverse of the case in primates [134]. Waxman and others have investigated clearance of IC via RBC [135–137], cited because of relevance to clearance of IC by platelets via their Fc receptors with consequent PMP shedding, as further reviewed below (see Section 4.3).

4. Modes and mechanisms of PMP production

3.10. Synthetic liposomes as model PMP 4.1. Introduction At least some properties of PMP may be investigated by synthetic liposome models. Most such studies have focused on PF3 activity and interaction with the complement (C) system. Chonn et al. have shown that negatively charged liposomes activated C via the Ca2 + dependent classical pathway, while positively charged liposomes activate C by the alternative path, and neutral liposomes had no effect [128]. However, an earlier report found that PS-containing vesicles (anionic) activated the alternative pathway (involving C3 not C1q), independent of antibody, and suggested that exposure of PS in senescent RBC could be a mechanism of their clearance [129]; PS exposure in platelets or PMP might activate C in the same manner (see also Section 4.3 ff). In a recent investigation of the rate of clearance of vesicles of known compositions, Chonn et al. showed that clearance rates in mice did not depend solely on charge but on the quantity of plasma proteins bound, especially ‘‘immune opsonins’’, and that this varied with lipid composition despite similar net charge [130]. In a similar vein, Xu et al. found that rat chyle chylomicrons could induce platelet aggregation, but only if pre-treated with plasma [131]. Citing earlier proposals by Bangham (1961) and by Papahadjapoulous (1962), to the effect that negatively

There is wide agreement that platelet activation is a necessary (but not always sufficient) condition for PMP production. Granting that, then the study of PMP production consists largely of the study of platelet activation, a broad topic extensively reviewed [138–143]. Because PMP generation is so tightly coupled to platelet activation, we briefly review that topic with emphasis on relevance to PMP.

4.2. By classical agonists Warren and Vales used TEM to study the morphology of platelet pseudopods arising from stimulation by surface adhesion or by collagen and observed the release of PMP [144]. It was shown by George et al. that thrombin causes production of abundant PMP, identified as platelet-derived by an immunoelectrophoretic assay for GP IIb/IIIa and visualized in TEM as heterogeneous submicron vesicles [145]; they also found the concentration of PMP in serum to be 10-fold that in normal plasma, implying that PMP are produced in the course of clotting. Early studies on production of PF3 activity by various platelet agonists are cited in [145], of

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more than historical interest. Also ca. 1982, Polasek made similar observations and developed special methods for SEM of PMP [146]. It was shown by Sandberg, Bode et al. that exposure of normal platelets to collagen caused a 7-fold increase in PF3 and PF1 (factor V-like membrane associated) activity, and that 41 and 31% of these activities, respectively, remained in the mediumspeed supernatant; TEM of the high speed pellet revealed two populations of particles, 80 – 200 and 400 – 600 nm [147,148]. It is now well-known that most platelet activating agonists, including ADP, thrombin, collagen, and calcium ionophores such as A23187 or ionomycin, can all elicit PMP production but with varying potency, usually listed in the following sequence [35,40,149]: epinephrine B ADP Bthrombin Bcollagen B (thrombin +collagen) B A23187 The wide differences in PMP produced by these agents, even at concentrations that are saturating with respect to aggregation, shows that the phenomenon of platelet activation is not a simple all-or-none phenomenon. However, many or most agonists can be additive or even synergistic [150]: a subthreshold level of one may greatly reduce the activation threshold of a second. For example, H2O2 released by neutrophils is insufficient to activate platelets but greatly reduces the activation threshold to arachidonate [151]. Hence even a weak agonist (such as epinephrine, elevated in stress [152]) may dangerously potentiate the action of others that would be harmless alone. In another example, activation by C3a was synergistic with ADP [153] (but not in washed platelets). Therefore, more sensitive assays of platelet preactivated status may be clinically important. This status may resemble the ‘‘primed state’’ of neutrophils [154], but may be temporary since platelets may recover, reverting to a resting state (as by active extrusion of Ca2 + ) or even to a refractory state: the shedding of PMP to carry off activation-prone membrane complexes (e.g. fixed complement) is commented in Section 4.3. A few examples of platelet activation relevant to PMP are given following.

4.2.1. Platelet acti6ating factor (PAF) Interest in the role of PAF [155] in thrombosis [156,157], thrombocytopenia [158], and inflammation and anaphylaxis [159] has been growing in recent years, and many studies investigating PAF receptor antagonists for potential therapeutic applications have appeared [160 –162] and may soon join the ranks of antiplatelet therapies. PAF has been shown to exhibit significant and synergistic actions on platelets down to picomolar concentrations [163]. Of special interest here is the recent finding that 87% of the PAF released by platelets was bound to the PMP [72]. The half-life of various PMP species in circulation has not been addressed.

4.2.2. Vasopressin, DDAVP Vasopressin, aka antidiuretic hormone (ADH), has at least one type of specific receptor on platelets [164,165] and is a potent platelet activator. A synthetically modified form, desmopressin (DDAVP), has found wide clinical application as an anti-hemorhaggic agent, particularly in hemophilias and von Willebrand’s disease [166,167], but increasingly also in other bleeding disorders, apparently independent of its known action in releasing vWF from the vascular endothelium [168], for which no rational explanation was available since it appeared to have no direct action on platelets [169]. However, it is now known that DDAVP does act on platelets, causing a rise in Ca2cyt+ and release of PMP both in vitro and in vivo, and corresponding elevation of PF3 activity [170]. These and other findings were overlooked in a recent review [171] emphasizing clinical applications. Although these effects are weak, they may be sufficient for restoration of hemostatic competence, and/or to sensitize (prime) the platelets, rendering them more responsive to physiological hemostatic forces arising in surgery. It has been shown that cryoprecipitate from donors treated with DDAVP is greatly enriched in PF3 and FVIII [172], probably attributable to promotion of PMP production by DDAVP, further supporting the hypothesis that promotion of procoagulant PMP and elevated Ca2cyt+ is a major mechanism of its vWF-independent hemostatic benefit. 4.2.3. Plasmin It has been shown that PMP functionally associate with fibrin at a thrombus [173] and thus may participate in fibrinolysis, as do whole platelets [174,175] including by inhibition [176]. There has been debate about whether plasmin inhibits or activates platelets; see refs in [177,178]. A more recent report concludes it does neither but potentiates prothrombinase formation, ‘‘probably by increasing the availability of factor V…’’ [179], again suggesting involvement of PMP. Plasmin exerts a dual effect on Ca2cyt+ [180]. Kinlough-Rathbone reported that plasmin inhibits platelet response to thrombin, but sensitizes platelets to other agonists [181]. The report of Braaten et al. is also suggestive in these regards [182]. 4.3. PMP by complement (C) in absence of antibodies There is reason to believe that C activation may be a major cause of PMP elevation in some clinical disorders. In addition to its immune function, the C system is intimately involved with the coagulation system and plays important roles in hemostasis [183,184]. Blood cells are normally equipped with specific GPI-anchored membrane proteins (DAF, CD59, others) which inhibit C activation and protect them against C-mediated

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damage [185]. If not arrested, C activation at the cell membrane leads to activation, vesiculation, lysis and cell death. These reactions may be promoted by Cfixing Abs (see Section 4.4), by immune complex (IC), and in other ways. Pinckard et al. in 1973 described activation of C by exposure of plasma to heart subcellular membranes [186,187], identified as mitochondrial and species-specific, recently further elucidated [188]. As mentioned in Section 3.10, PS-containing liposomes exert a similar effect, as can PMP (see below). Polley and Nachman in 1983 described platelet activation by direct incubation of gel-filtered platelets with C3a, which was synergistic with ADP and localized to C3a des-arg peptide, suggesting to the authors the existence of a specific C3 receptor on platelets [153]. Peerschke et al. identified a C1q binding site on platelets [189]. The binding of C3 to platelets, often correlated with anti-platelet Abs in ITP, has long been of interest (for example, [190]) though its significance remains obscure. Platelets contain and can secrete C5 – C9 [191]. Sims, Wiedmer, Esmon, and colleagues have extensively investigated platelet activation and associated PMP production caused by exposure of platelets to purified C components. Using a dye to investigate changes in membrane potentials of gel-filtered platelets, they found in 1985 that C5b-9 caused rapid depolarization followed by repolarization over 15 – 20 min; the repolarization was blocked by oubain or by using a buffer with K + instead of Na + [192]. They conclude that in vivo, this mechanism is most likely in the circmstance of autoimmune activation of complement (final sentence), and suggest that recovery of membrane potential may follow the shedding of PMP, which carry off the C5b-9 pore (membrane attack complex, MAC). That suggestion was confirmed in [9]; reference to a similar effect in neutrophils is given. The process was dependent on external Ca2 + , presumably admitted through the C5b-9 pore (for debate on the nature of this pore, see [193]). Similar findings have since been reported for RBC vesicles by Iida et al. in 1991 [194] and by Pascual et al. in 1993 [195]. It thus appears that one function of the shedding of PMP/EMP is a rescue mechanism. Butikofer et al. showed with RBC that GPI-anchored membrane proteins, including DAF, are enriched in the shed microparticles [8]. Wiedmer et al. demonstrated in 1986 that PMP released by exposure to either A23187 or C5b-9 in the presence of Ca2 + exhibit procoagulant activity, serving as a site for the assembly of the prothrombinase complex [196,197]. They further characterized the PMP, showing the presence on them of GP Ib, GP IIb/IIIa, and the a-granule membrane protein GMP-140 (now CD62p, P-selectin), in addition to great enrichment of C5b-9 [198]. An activation-dependent epitope of GP

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IIb/IIIa recognized by the mAb, PAC-1, was found on C-activated platelets but not on PMP. They found fully half of the total factor Va expressed from the a-granules associated with PMP, and concluded that ‘‘the [PMP] shed by C5b-9 treated platelets (and not the platelets themselves) provide the principle binding sites for coagulation factor Va and serve as the principle catalytic surface for the prothrombinase complex’’ [196]. Objections to this and related claims are discussed in Section 4.7. They also investigated the role of Ca2 + and calpain in C5b-9 induced vesiculation [199], subsequently extended to the role of protein kinases [200]: Ca2cyt+ was increased by C5b-9, regardless of inhibitors of protein kinases or phosphatases; in fact, vanadate increased PMP production; but PMP were forestalled by inhibitors of the kinases or of calmodulin, which ‘‘suggests a role for the platelet myosin light chain kinase or another Ca2 + /calmodulin-regulated membrane component’’. In 1991, they gave evidence that PMP can express high-affinity receptors for factor VIII, described as ‘‘a cofactor in the tenase complex which assembles on the membrane of activated platelets’’, and showed that the avidity of platelets for FVIII quickly falls off while that of the PMP is sustained [201]. For summaries on those studies of the interaction of C with platelets, see [202–204]. In 1993, the same group further examined the role of the 18 kDa inhibitor on the potency of action of C5b-9 cited above [205], having by then recognized it as CD59 (along with CD55, aka decay acclerating factor, DAF) [15], the former having been isolated from platelets by Morgan in 1992 [206], the latter having a longer history because of wide interest in paroxysmal nocturnal hemoglobinuria (PNH). Patients with PNH were selected for study because of their now well-known defect in GPI-anchored membrane proteins: as might be expected, a 10-fold increase in sensitivity to C5b-9 was seen, as measured by prothrombinase activity and PMP generation [15]. We have confirmed the much greater sensitivity of PNH platelets to C-mediated damage, and have also observed, using an mAb to CD59 kindly supplied by B.P. Morgan, that a subset of ITP patients have platelets deficient in CD59, which deficiency correlated with sensitivity to C-mediated damage [207].

4.4. PMP by anti-platelet Ab with/without complement (C) Since the introduction of mAbs, many new insights on interactions of platelets with anti-platelet Abs, often leading to PMP shedding, have come to light. Rubinstein et al. [208] distinguish between murine mAbs which cause platelet activation independent of C vs. dependent on C, referring to the latter as lytic and noting that two of nine mAbs with this property were

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of IgG2a subclass and one was IgG3 [209,210] (the potency of human IgGs in C activation is elsewhere given as IgG1 : IgG3 \IgG2 IgG4 [211].) Although findings with murine mAbs apply to human antibodies (Abs) only with reservations, important insights have been gained from them, and Deckmyn comments that human platelet-activating Abs ‘‘seem to parallel the mode of action of certain murine antibodies’’ [212]. Nomura et al. reported two mAbs recognizing platelet FcR (FcgRII) which induced C-dependent aggregation, and commented that these mAbs ‘‘may be useful to study the relationship between autoantibodies, the Fc receptor, and complement’’ in ITP [213]. On the human side, Honda et al. found two C-fixing auto-Abs in an ITP patient, both IgM, and suggested platelet destruction via C3b-receptor mediated phagocytosis [191]. Cines et al. reported anti-platelet IgM in 57% of ITP patients, that it correlated with amount of bound C3, and that C may play a major role in platelet destruction in ITP [214]. Similarly, Nomura et al. found that PMP shedding from normal washed platelets was induced by 12 of 56 ITP plasmas and implicated IgM [215]. Immune complex (IC) can also lead to C-mediated platelet damage, e.g. [216,217]. We chanced to rediscover murine mAb-associated C-mediated platelet lysis while trying out a then-new marker of GP IIb/IIIa for flow cytometry, PLT1 (Coulter Corp., prepared free of gelatin to avoid false PMP), an IgM, which promised a brighter FITC signal for PMP detection, but were dismayed to find that it caused a time-dependent rise in PMP and a decline in platelets (lysis), effects which were abolished by heating the plasma to block C activation [14]. Those observations led us to notice that other mAb platelet markers also exerted such effects, though less dramatically, implying a need for caution in interpreting PMP measured in whole blood or plasma by anti-platelet mAbs, because of factitious C-mediated PMP production. Accordingly, we now measure PMP only in platelet-poor plasma with F(ab%)2 mAb [14,36]. These findings suggest that at least some auto-Abs in ITP may cause C-mediated platelet destruction and PMP production, since this hypothesis would help explain why some but not all ITP patients exhibit elevated PMP ([13,14] and see Section 5.2.2). C-mediated platelet lysis has been documented in severe refractory immune thrombocytopenia [218]. We found that phagocytosis of opsonized platelets by U-937 cells does not cause shedding of PMP (unpublished). Therefore, high PMP and/ or PF3 activity may be a marker of C-mediated platelet lysis. Complement was required for activation of platelets by high-titer anti-phospholipid Abs (aPL) [102]. Approximately 30% of ITP patients were found to have PL/aCL [219,220]. Kiefel et al. [221] cited two ‘‘rare exceptions’’ of C-mediated lytic destruction in ITP, and other thrombocytopenias where this mechanism pre-

dominates (e.g. drug-associated, alloimmune against HLA). C-mediated platelet lysis may also be detected by measuring available C [222], or C activation [223], or PMP enriched in C (because of the findings of Sims et al. in Section 4.3). The role of C in ITP was prominent in earlier literature [224–229]. Nomura et al. suggest that active phagocytosis can be detected by CD68 positive microparticles [17]. The problem of determining the mode of platelet destruction in ITP is of immediate clinical import because different treatment strategies are called for, as remarked in [14]. Solumn et al. show that the protease inhibitor, leupeptin, inhibits Ab-mediated platelet destruction by MAC [230]. However, despite extensive research, there is yet no consensus on these questions or on the mode of PMP production in ITP.

4.4.1. Platelet acti6ation/PMP generation by Abs independent of C Among the many clinical settings in which PMP are elevated, the first and best documented is in ITP, where anti-platelet Abs interact with platelet-specific Ags [25,231] (see also Section 5.2). It was established by Harrington et al. in 1959, by means of self-experiment, that ITP is caused by anti-platelet Abs [232]. Colman et al. showed in 1977 that anti-platelet rabbit serum could induce platelet aggregation and release, and that these reactions occurred even if the serum had first been heated (56°C, 30 min) to inactivate C [233]. The emerging consensus on such effects is summarized briefly: (i) Murine mAbs that activate platelets independent of C are of the IgG1 subclass [234]; (ii) nearly all target one of just three antigens, CD9 (GP 24), CD36 (GP IV, aka GP IIIb), CD41 (GP IIb/IIIa) [234]; (iii) activation requires the Ab bind also to FcgRII (‘scorpion model’) [208,235]; (iv) these events often or always involve inter-platelet agglutination (below) and (v) result in clustering (or ‘oligomerization’) of the receptors [235,236], akin to the patching and capping seen in neutrophil activation, now a recognized feature of platelet activation [237–239]. Some or all of the usual signal transduction events [143] then eventuate [208] but details remain controversial [240]. 4.4.2. Importance of epitope Not all Abs against these antigens necessarily promote platelet activation and PMP production. For example, mAb IV.4 against FcgRII is widely used to block activation by FcgRII liganding, but another antiFcgRII, CIKM5, causes activation [208]. Similarly, Yanabu et al. identified two human auto-Abs against CD9 in an ITP patient, one of which activates while the other blocks the action of the first [241]. Other Abs are apparently neutral: Bak (aka Lek) Abs, which target GP IIb, are without observed effect [242]. These observations raise questions about the clinical value of identifying target antigens in ITP. Many human auto-Abs are known to blunt rather than promote normal platelet

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functions, notably, some against GP IIb/IIIa [212,243], probably accounting for serious bleeding in ITP patients whose thrombocytopenia is only mild. Yanabu et al. found that 12 of 60 ITP patient Abs inhibited ADP-induced aggregation and 11 of these were anti-GP IIb/IIIa [244].

4.4.3. Immune complex (IC) Platelets may also be activated by capture of IC or aggregated IgG by FcR. FcR density on platelets is variable in individuals and between individuals, and some is apparently internal, since thrombin stimulation causes a  50% increase in its surface expression [235]. The normal function or raison d’etre of platelet FcR is a matter of speculation, but clearance of IC is probably not it, since, as earlier remarked (see Section 3.10), RBC are believed to be the main mechanism of IC clearance. A plausible scenario tendered in [208,235] is that IC in the vicinity of a wound could locally activate platelets, aiding in their recruitment to an inflamed site. 4.4.4. Mechanism of FcR acti6ation Exactly how FcR liganding triggers the biochemical machinery of activation is uncertain, but clues are found in platelet activation associated with vWF liganding to GP Ib-V-IX, said to be highly sensitive to shear-induced platelet activation (SIPA) [239], implying triggering by torque of the tree-like structure by stress on its cytoplasmic ‘roots,’ connecting to filamin, actin, etc., since it is known that ‘‘actin filaments [are] linked by actin-binding protein’’ to GP Ib [78]. The same may be true for FcR, known to have similar connections [245], since cross-linking of FcR, which also causes activation, implies similar torque-induced stresses. 4.4.5. Platelet agglutination Abs can cross-link platelets, causing agglutination (as for RBC in Coomb’s test), but in platelets this may cause activation and PMP release. Horsewood et al., Rubinstein et al., and Anderson et al. used elegant approaches to investigage inter- vs. intra-platelet Abmediated activation, e.g. preparing two batches of platelets, one loaded with 14C serotonin but with access to Ag blocked by F(ab%)2 fragments, the other fixed with formaldehyde, bearing intact Ab and with FcR blocked by mAb IV.3, then mixing them: activation ensued, judged by release of serotonin, proving Plt – Plt interaction in activation by the given Ab [208]. Platelet aggregates (PAg) classically arise independently of Ab. Their occurrance in pathology and formation in vitro following stimulation by agonists has been studied by Ahn’s group [246,247] (and by others cited therein), with the aim of developing clinically practical assays of prothrombotic states. Certain mAbs

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can trigger fibrinogen-mediated PAg without addition of exogenous agonist, e.g. by mAbs D3GP3 (anti-GPIIIa) and D33C (anti-GPIIb), as cited in [248]. In view of platelet activation by Ab-mediated interplatelet cross-linking, it seemed that such agglutination might be a necessary requirement. This was claimed from studies of dextran gel-immobilized platelets [249]; but another study with agarose-immobilized platelets concluded that both mechanisms could work, reviewed in [250]. A possible objection to studies with gel-immobilized platelets is that the gel might also restrict platelet surface receptor mobility (clustering). Thus Worthington et al. used diluted platelets to minimize interaction, and concluded that, although most activating mAbs required inter-platelet interaction to induce secretion, intra-platelet Ab binding could also induce activation with certain Abs, as cited in [208].

4.4.6. PMP and GP IIb/IIIa Abs Although methods of identifying target Ags in ITP remain controversial, epitopes of GPIIb and GPIIIa (or their complex) appear to be the most common [251]. As noted above, these Abs often inhibit platelet function. If and when platelet activation occurs by Abs to GP IIb/IIIa (in absence of C), it is presumably FcR-dependent. Reverter et al. found that the chimeric Fab fragment c7E3 (ReoPro™), which blocks fibrinogen binding to GP IIb/IIIa, inhibited PMP production by thrombin but the intact Ab 10E5 did not [18] (we found in unpublished work that c7E3 did not prevent CD62 expression by ADP). Karpatkin et al., in a somewhat puzzling report [252], found no binding to GP IIb/IIIa of F(ab%)2 IgG from 39 ITP patients, and that positivity in their hands depended on aggregated IgG, as may form in the frozen state. They suggested that immune complex (IC), not antigen-specific Ab, is the cause of ITP. That hypothesis dates to 1977 and was addressed by Kiefel in 1986 [253] who concluded that ‘‘our data render the role of IC in the pathogenesis of ITP very questionable’’. Subsequent to Kiefel’s MAIPA assay of 1987 [254], leading to a plethora of others, e.g. [255,256], the evidence has become overwhelming that ITP does indeed yield platelet antigenspecific Abs, as indicated above, where F(ab%)2 fragments are routinely used as probes. However, the more conservative closing words in [252] are doubtless good advice: ‘‘We propose that the presence of true antiplatelet antibody should be evaluated in nonstored, recently prepared serum from nontransfused patients... [and that] stored sera (or IgG) should be gel-filtered to remove high molecular weight fraction...’’. 4.5. PMP in blood-bank products; platelet storage lesion PMP arising in blood-bank platelet concentrates and cryoprecipitates has been extensively studied. The

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‘‘platelet storage lesion’’ has been recognized as a serious problem in transfusion medicine since 1960 [257]. The shedding of vesicles from stored RBC is also of continuing interest [258] but platelets are more perishable, unique in being activated by chilling, which induces shape change, release, increased surface expression of IgG, shedding of PMP [259,260] and loss of GP Ib [261]—or that portion of it called glycocalicin, found on shed PMP [262]. Platelet activation and PMP generation by chilling may be attributable to effects of cold on microtubules [263,264]. Following early observations by Wolf [3], PMP and associated PF3 activity in platelet concentrates were characterized in the 1980s by Sandberg, Bode et al. [147,265]. In 1986, they reported further study of blood banked plasma, employing a then-novel PF3 assay based on a chromogenic substrate for thrombin, finding that by 72 h, 54% of the PF3 activity was in the supernatant but the platelet number was not diminished [40]. At about the same time, George et al. investigated PMP in cryoprecipitates, measuring PMP in the platelet-free supernatant (PFP) and in the high-speed ‘‘invisible pellet’’ by 121I-labeled mAb to GPIIb, finding high levels of PMP in the cryoprecipitate [10], implying that the hemostatic benefits of cryoprecipitate in bleeding disorders may be attributable in part if not entirely to PMP. In a similar vein, Solberg et al. measured PMP in blood bank platelet concentrates using a modified Coulter Model D blood cell counter, and found a clear correlation between PMP and PF3 activity [26]. They also noted a correlation between increasing PMP and alkalinity of the stored platelet concentrate, both rising in parallel with free LDH as surviving platelets declined; in view of these findings they warn of ‘‘clear dangers’’ of thrombosis in patients receiving platelet concentrates with high PMP [145]. On the other hand, it may be added that cryoprecipitate is often used for the express purpose of augmenting clotting in patients with bleeding disorders [266], similar to the rationality of DDAVP (see Section 4.2.2). It is likely that even cell-free plasma contains procoagulant PMP, since Rock et al. find evidence of ‘‘important, and indeed, essential’’ platelet constituents in it [267]. In 1991, Bode et al. studied additives to limit damage to stored platelets, finding that activation inhibitors PGE1, theophylline, aprotinin, caused 40% reduction in PMP, 84% less PF3 activity, and 61% less LDH released [11]. A similar strategy was more recently investigated [268]. The correlation coefficient found in [11] between PF3 and the larger PMP was r =0.748. Most PMP were positive for GPIIb/IIIa (73%) and/or GPIb (43 – 46%) [10] but in their Discussion they speak of ‘‘variable losses’’ of GPIb on PMP, important because GPIb is often the marker used to quantitate PMP. Bode et al. also reported a rise in plasmin and activated C in stored platelets [269].

4.6. PMP arising by shearing stress, foreign surfaces In normal circulation, platelets are continuously subjected to varying viscose shear, another circumstance which, as known since the 1970s [270–273], modifies the behavior of platelets as compared to quiescent observations in vitro [274,275]. Interest in the topic of shear-induced platelet activation-aggregation (SIPA) may be gauged from the fact that a Medline search of the literature 1992–1996 yielded some 200 titles. For review as of 1985, with emphasis on comparative experimental methods, see [276]; for 1996 review, see Kroll et al. [277] and comment on it by letter on the role of RBC [278]. We include effects of foreign surfaces because much attention has focused on hemodialyzers and coronary by-pass machines, where abnormal shearing forces may combine with foreign-surface effects to activate platelets [279,280] and C [281], with pathological consequences discussed later (see Section 5.2.7), where it is argued that elevated PMP are particularly suspect. It may be added that the ease of platelet activation ex vivo may be due not only to foreign surfaces but also to the absence of platelet passivating substances normally present on, or continuously secreted by, the vascular endothelium, chiefly EDRF/ NO, eicosanoids (PGI2), and ecto-ADPase/CD39, an apyrase [282,283]. PMP formation during passage though such machines is often taken as a measure of adverse (procoagulant) effects. For example, Gemmell et al. adopt PMP as one of two criteria of adverse surface effects [284], along with platelet binding to leukocytes (see Sections 3.9 and 5.3). Similarly, Dewanjee et al. collaborated with Ahn’s lab to show in a porcine model a pronounced rise in PMP with time of circulation through a hollow-fiber hemodialyzer, which paralleled the formation and growth of thrombi [285]. Miyazaki et al. recently addressed mechanisms involved in PMP production by SIPA [101]. Complement (C) activation may be involved [281]. Such effects are not limited to extra-corporeal devices. Holme et al. has recently shown, using non-anticoagulated blood directly from an antecubital vein to a constricted perfusion chamber, that stenosis can cause platelet activation and PMP release [286]. Anecdotally, we have observed extremely high PMP in a patient with a severe mitral valve stenosis but no other known pathology. The effects seen by Holme et al. [286] occurred only at very high shear rates; but platelets become more sensitive to SIPA once vWF is liganded, as cited above (see Section 4.4). Siljander et al. studied PMP under controlled shear conditions in blood lightly anticoagulated with PPACK, a thrombin inhibitor, flowing over a collagen-coated surface, and determined that PMP played a major role in the formation of thrombi [173], chiefly via interaction with fibrin. SIPA

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is potentiated by DDAVP [287], consistent with comments above (see Section 4.2.2).

4.7. Voice of dissent An often-cited 1993 paper by Swords et al. [24] has lent credence to skeptics who doubt if PMP have any hemostatic relevance. For example, Ruggeri in an editorial [288] on [125] cites Swords et al. as casting doubt on the hemostatic relevance of PMP. Briefly, blood was collected in ACD with heparin (50 U/ml) and the platelets were washed in buffer containing 50 U/ml heparin. Flow cytometry of platelets and PMP was by both light-scatter and FITC-labeled mAb against GPIIb/ IIIa, and mass-percent of PMP released was estimated by comparing the size and number of PMP vs. platelets. By these methods, the adhering platelets did not release significant PMP or prothrombinase activity to the supernatant following addition of thrombin. They concluded that ‘‘…microvesicle release was not essential in the expression of platelet procoagulant activity. In the case of adherent platelets, this raises questions as to the validity of reports suggesting a link between platelet activation, release of microvesicles, and expression of platelet procoagulant activity’’. This conflicts with findings dating back to 1972 [5]. More recently, Siljander et al. specifically addressed the conclusions in [24] and reached an opposite conclusion, confirming a role of PMP [173]. Heemskerk showed blebbing of platelets adherent to collagen [103]. A possible explanation for the negative result of Swords et al. is their use of high levels of heparin, known to diminish or abolish platelet responses [289,290]. De Groot and Sixma showed that completely opposite results can be found with heparin vs. other anticoagulants [291]. In sum, the evidence given in [24] is not persuasive support for its conclusions, and we are left wondering why that single paper has been so widely and uncritically accepted.

5. Clinical relevance The studies reviewed above are highly suggestive of important roles for PMP in hemostasis and thrombosis. More direct clinical evidence for such roles has emerged in recent decades.

5.1. Deficient 6esiculation: Scott syndrome and related defects 5.1.1. Scott syndrome Although several defects in PF3-like procoagulant activity have been described [292 – 294] (and see references in [295]), a rare bleeding disorder, Scott syndrome, was described in 1979 [296] which bears on questions about the mechanism and clinical relevance of membrane

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asymmetry/flip-flop and PMP in platelet activation. It was shown in 1989 that platelets from patients with Scott syndrome were ‘‘markedly impaired in their capacity to generate microparticles [PMP] in response to all platelet activators, and this was accompanied by a comparable decrease in the number and function of inducible factor Va receptors’’ [297]. In 1992 that work was extended to Scott erythrocytes (E), showing that like Scott platelets, Scott E yielded B10% of the prothrombinase activity of normal E following treatment with A23187, and remained discoid as seen in SEM while normal E became echinocytes [298]. This was also observed in resealed E ghosts, showing that ‘‘the defect was intrinsic to the membrane structure’’. Scott syndrome was reviewed by Weiss in 1994 [299]. Later, another family was found and it was reported in 1996 that Scott syndrome is ‘‘transmitted as an autosomal recessive trait reflecting the deletion or mutation of a putative phosphatidylserine translocase’’ [300,301]. A more recent report shows that this translocase (scramblase, see Section 3.7) is present but inactive [302], probably because of a defect in signal transduction [303].

5.1.2. In6erse Scott syndrome Stormorken et al. reported on a bleeding tendency associated with ‘‘a complicated syndrome’’ (Stormorken’s), termed an ‘‘inverse Scott syndrome’’ since in this case the platelet procoagulant activity was fully expressed even without deliberate stimulation; i.e. the normally in-facing PS is evidently out-facing or scrambled in the resting platelets of this syndrome [304]. Such a case would be expected to result in a thrombotic disposition but in fact there is a bleeding diathesis, a circumstance the authors call ‘‘paradoxical’’, perhaps because of ‘‘clear evidence of significant spontaneous microvesiculation in these patients’ blood’’ [304]. They speculate on explanations, including unusual response to shearing stress. We are beginning to appreciate that such seeming paradoxes are commonplace, e.g. PMP may be procoagulant or anticoagulant (see Section 3.8); that high PMP do not always imply thrombotic disposition (see Section 5.1); that ITP may be associated with thrombosis as well as bleeding (see Section 5.2); that anti-PPL Abs such as the lupus anticoaglant (LAC) are often associated with thrombosis (see Section 5.2.5); that plasmin has dual and opposite effects (see Section 4.2); etc. 5.1.3. Castaman’s defect Castaman et al. reported another inherited bleeding disorder, found in four probands with prolonged bleeding times, in which PMP production is impaired (18–25% of normal response to agonists), yet differs from Scott syndrome in having normal prothrombinase activity [305]. Although the patient serum had abnormally high levels of residual (unconverted) prothrombin, as in Scott syndrome, unlike Scott syndrome addition of commer-

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cial PPL did not correct the abnormality in a prothrombin consumption test. The authors suggest that the evidence implies an isolated defect specific for PMP formation in these patients.

5.2. Abnormally ele6ated 6esiculation (PMP production) 5.2.1. Introduction The presence of PMP in a clinical disorder was first described in ITP [25,231]. They observed PMP and EMP in patients with ITP by transmission electron microscopy (TEM). However, quantitative studies are unreliable by TEM. In 1986, quantitative elevation of PMP was documented by flow cytometry in patients undergoing cardiopulmonary bypass (CPB) during extracorporeal circulation and in patients with acute respiratory distress syndrome (ARDS) [53]. Many additional clinical disorders have since been associated with elevated PMP, reviewed following. 5.2.2. PMP in immune thrombocytopenic purpura (ITP) (see also Section 4.4) PMP in the setting of ITP was investigated by flow cytometry in 1991 [215]. Elevated PMP in ITP was confirmed in 1992 [13]. Of particular interest was the finding in the same report that ITP patients who were asymptomatic of thrombocytopenia had about 2-fold higher PMP than those with symptoms, suggesting that PMP in ITP is hemostatically functional, apparently protecting patients from bleedings. It is well-known that platelet count alone is not a reliable indicator of hemorhaggic risk: some patients with severe thrombocytopenia do not bleed while others with higher platelet counts bleed extensively. PMP assay may be a step in the direction of better predicting bleeding risk. On the other hand, the correlation between high PMP and freedom from bleeding in ITP is only statistical [13], meaning there are exceptions. These exceptions may be attributable to heterogeneity of PMP arising from different mechanisms, different status of platelet activation, efficacy of compensatory mechanisms of hemostasis, or to the status of endothelial integrity. Equally interesting was the finding in the same paper of a group of patients with unusually high levels of PMP who were experiencing subtle symptoms of transient ischemic attacks: episodic dizziness, blurred vision, numbness and tingling of extremites, facial palsy, dysarthrias, imbalance of gait, confusion [13]. Brain CAT scans of some of the cases showed small-vessel thrombosis such as lacunar infarcts, and MRI often revealed patchy subcortical lesions consistent with ischemic small vessel disease. These findings implied that PMP in some ITP patients may be thromobogenic, contributing to small-vessel thrombosis, particularly in the CNS. Followup of these patients often revealed gradually progressive cognitive dysfunction leading to dementias [13].

Why are PMP often elevated in ITP? Beyond earlier comments (see Section 4.3 et seq), some insight on this question was gleaned from in vitro studies employing two mAbs reacting to platelet specific antigen CD41 (GP IIb/IIIa), one of IgG class and one of IgM class: PMP generation correlated with concentration of mAb and was largely abolished by preheating plasma to inactivate complement (C), indicating involvement of C in Ab-mediated PMP generation [14]. With the IgM (PLT-1), which fixes C more efficiently, platelets were often rapidly lysed (undetectable by flow cytometry), even though procoaglant activity measured by RVVT continued to rise. These findings and others (see Section 4.4) suggested to the authors that some antiplatelet Abs can induce C-mediated platelet destruction and PMP generation.

5.2.3. PMP in transient ischemic attacks (TIA) In view of their observation of small-vessel TIA in patients with ITP and high PMP, the same group undertook PMP assays of non-ITP patients with cerebrovascular accident (CVA) or other neurologic disorders. Four patient groups were studied: large vessel strokes, small vessel strokes (lesions B 2 cm by CAT scan), multiinfarct dementias, and Alzheimer (AZ). All except AZ had significantly elevated PMP [12]. Levels of PMP were higher in small-vessel strokes (small-vessel TIA, lacunar infarcts) than in large vessel strokes. It thus appears that elevated PMP are associated with small-vessel thrombosis, with or without ITP. Granting that PMP are preferentially associated with small-vessel CNS disorders, it may be relevant that the CNS vascular endothelium is unique in lacking thrombomodulin [306,307]. Perhaps relatedly, a standard bleeding time (BT) test appears to be independent of CNS bleeding tendencies, since aspirin markedly prolongs the skin BT in an animal model but brain bleeding time was unaffected [308]. Brain endothelium is also partly deficient in neutrophil adhesins [309]: PMP associate with neutrophils [125] and have been implicated in monocyte adhesion to endothelium [126]. Activated platelets have long been implicated in ischemic stroke [310,311], supported experimentally in rabbits [312] but in our experience, elevated PMP are associated chiefly with small vessel occlusions, manifesting as small vessel TIA, mini-strokes, or progressive cognitive dysfunction leading to dementias. Nomura et al. reported that PMP rich in amyloid b-protein precursor (APP) sharply discriminate between normal controls and patients with cerebral infarcts or diabetes (PB0.001) or uremia (PB0.01) [73]. They found that the correlation of APP-positive PMP with cerebral infarcts is superior to that for undifferentiated PMP, implying that APP positivity reflects a subset of PMP exhibiting enhanced correlation with thrombosis. Incidentally, abnormal platelet-associated APP has also been found in Alzheimer’s disease (AD) and Down’s syndrome [313].

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In view of the small-vessel diseases common in diabetes, it would not be surprising if PMP were implicated there too; indeed, Rao et al. described platelet abnormalities in diabetes mellitus [314], and see also [315].

5.2.4. PMP in acute coronary syndromes (ACS) A further indication of thrombogenic PMP stemmed from a case study of a young cardiologist with mild ITP but elevated PMP, who suffered acute MI despite few known risk factors [316]. Data on 12 similar cases (ACS at young age without known risk factors or CAD) showed chronic platelet activation by a suite of indicators consisting of mild thrombocytopenia, increased PMP, elevated platelet-associated IgM, and enhanced procoagulant activity of plasma fractions [316]. All did well on antiplatelet therapy, since no recurrence of ACS was seen over a 3+year followup, and platelet activation markers (including PMP) improved or normalized. These findings suggested to the authors that chronic platelet activation can lead to ACS without atherosclerosis and that this subgroup was protected by antiplatelet therapy [316]. Chronic platelet activation may sensitize to ACS since many of these patients reported mental stress (which raises epinephrine) just prior to their acute episodes. Tate et al. reported increased PMP in coronary sinus blood from patients with ACS [317]. Katopodis et al. evaluated platelet calcium homeostasis and activation markers in three groups undergoing coronary angiography for suspected ACS: recent MI, unstable angina, and a patient control group (stable disease or non-coronary artery disease such as valvular heart disease or non-cardiogenic chest pain with normal angiography) [318]; PMP, CD62p (P selectin) expression, platelet– leukocyte interaction and platelet Ca2 + homeostasis were measured. PMP was significantly elevated in ACS compared to patient controls, but platelet – leukocyte interaction was increased only in unstable angina. PMP assay distinguished patients with UA or recent MI from those with stable disease or normal coronory arteries, at PB0.001 [318]. 5.2.5. PMP 6ersus PF3 acti6ity in thrombosis PMP may be heterogenous in potency of procoagulant (PF3) activity, some being anticoagulant by virtue of associated activated protein C (aPC; see Section 3.8). Jy et al. compared PMP counts with PF3 activity by RVVT (explained in Section 2.2) in groups of thrombotic patients: PMP were significantly elevated in thrombotic disorders, and the RVVTs were shortened in all three plasma fractions (PRP, PPP, and 0.1 mm filtered PPP) [36,319]. They showed that PMP \ 0.1 mm contributed most of the procoagulant activity in the thrombotic group but not in the normal group, and concluded that assay of PMP+ RVVT sharply discriminated thrombotic conditions (P B 0.001); conventional

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coagulation tests (PT, aPTT, fibrinogen) did not distinguish the thrombotic patients [36,319]. However, the RVVT correlated only marginally with PMP (P:0.05), whereas Bode et al. found a better correlation between PMP and PF3 activity (in stored platelets) [11]. Possible explanations were given above (see Section 5.2.2), to which may be added the fact that the PMP assay in [36] relied on antigen detection rather than direct detection by light scatter, meaning that some species of PMP may have escaped detection. If the PF3 activity of PMP is partly masked by aPC, timing of the assay relative to blood drawing may be important in view of the short half-life of aPC. At any rate, Jy et al. showed that the RVVT is a sensitive indicator of thrombotic disposition; they consider PF3 assay as complementary to PMP enumeration in assessing prothrombotic states [36]. It remains to be clarified if, or to what extent, PMP are a direct contributing cause of thrombosis, as distinct from being a relatively innocuous concommitant of platelet activation. The evidence that PMP are indeed hemostatic is most persuasive in ITP [13] since in that case whole platelets are largely absent. Since PF3 activity is increased in thrombosis, and since the main source of PF3 in plasma is PMP (barring RBC pathologies, etc.), then the obvious implication is that PMP are thrombogenic. Studies on Scott syndrome (see Section 5.1.1) tend to support this conclusion.

5.2.6. Antiphospholipid antibody (aPL), lupus anticoagulant (LAC) Anti-phospholipid Abs have been recognized as a risk factor for thrombosis since first described [320– 324]. A major advance has been the recognition that aPL are not usually directed against PPL as such, but against some PPL-bound cofactor antigen, chiefly b2glycoprotein-I (b2GPI, aka apolipoprotein H) but others are cited below. It is likely that aPL often go undetected owing to cofactors other than b2GP1, not usually tested for. b2GP1 copurifies with thrombospondin [325]. For reviews, see [326–331]. For reviews of diagnostic criteria and assay methods, see [331–333]. 5.2.6.1. Rele6ance to PMP. PMP have been proposed for improved assay of LAC [334]. Direct evidence for PMP involvement was given by Nomura et al., who showed that elevated PMP in ITP can express b2GPI, the major aPL antigen, and that this expression correlates with the presence of anti-cardiolipin (aCL) [335]. They conclude that aCL activate platelets in ITP and cause the generation of PMP rich in b2GPI and P-selectin. b2GPI binds to anionic PPL and inhibits prothrombinase activity of platelets and PMP [336], contact activation of coagulation [337], and serotonin release from platelets exposed to ADP [338]. As cited in

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Section 4.4, aPL are found in  30% of ITP patients, and conversely, platelet specific Abs are found in SLE [339].

5.2.6.2. Cytoplasmic Abs and PMP. Natural Abs against cytoplasmic proteins are increasingly recognized in recent years, including in aPL [339]. Hou et al. report ITP auto-Abs against cytoskeletal tropomyosin [340], and cite related reports. If PMP can occur as inside-out membrane fragments, then cytoplasmic antigenic domains would be exposed on their outer surface, possibly explaining at least some anti-platelet auto-Abs against cytoplasmic domains. Beer et al. showed that inside-out PMP can indeed exist [341], depending on mode of production (as with RBC ghosts or mitochondrial MPs). It appears that auto-Abs can sometimes penetrate into cells [342] (a familiar example being the anti-nuclear Abs, ANA), including some anti-platelet auto-Abs [343,344]. Exposure of normally sequestered antigens is believed to be one mode of inciting autoimmunity, different from cross-reaction/mimicry, etc. [345,346]. 5.2.6.3. Platelet acti6ation in aPL. Several authors reported aPL-induced platelet activation by the mechanism previously discussed (IgG cross-linking from target antigen to FcR), e.g. [347] and see below. Lin and Wang have shown that aCL raised in the rabbit are potent activators of human platelets, and in subthreshold quantities are synergistic with other agonists [348]. The F(ab%)2 fragment had no effect alone but blocked the action of the intact IgG; activation was not inhibited by mAb blockade of FcR. Such conflicting reports may be resolved in part by the finding of Stewart et al., that all sera wth high-titer aPL caused activation of the C system leading to platelet activation via the MAC complex [102]. This route of activation (see Section 4.3) might be overlooked in citrated blood, which limits Ca2 + and Mg2 + required for C activation; Stewart et al. used the thrombin-directed anticoagulant, PPACK. Kurata et al. proposed a method of distinguishing anti-platelet auto-Abs from immune complex (IC) in platelet activation in SLE [349]. According to Roubey’s review [328] ‘‘there is little evidence for [aPL] binding to fresh unstimulated platelets’’ (his refs. 154 – 157). 5.2.6.4. Other antigenic cofactors in aPL. The best known antigen cofactor of aPL is b2GP1 but many others are known. Oosting et al. argued that aPLs may be directed against combinations of PPL with prothrombin, protein C, or protein S [350], all of which have been documented to occur on at least some species of PMP. Protein S is sometimes inhibited by auto-Abs [351] (it may be relevant that protein S carries about 50% of the circulating C4b-binding protein (C4BP) [352]). They offer this as a possible explanation for the often conflicting clinical manifestations of aPL, stating that ‘‘the identity of the plasma proteins involved in the binding of aPL might

determine which pathogenic mechanism causes thrombosis’’ [350]. Roubey seems to concur, and lists other antigens [328], as does Schultz in his Table 3 [331]. Often overlooked in such lists of aPL antigens or ‘cofactors’ are annexins [353–358], also implicated in rheumatoid arthritis [359,360] and recently shown important in pregnancy loss [361]. Tokita et al. have shown a ‘‘specific cross-reaction’’ of aPL IgG with platelet GP IIIa [362]. Sorice et al. argue that aCL and anti-b2GPI are distinct antibodies [363], a controversy addressed in [328,331], the resolution of which appears to be that both probably occur, i.e. ‘true aCL’ is said to ‘‘occur in the setting of syphilis and other infectious diseases’’ [328]. A variety of methods for distinguishing antigenic cofactors have been given, as in [364], and see [331].

5.2.7. Additional e6idences of PMP in thrombotic disorders 5.2.7.1. Thrombotic thrombocytopenic purpura (TTP). Kelton et al. found high calpain in sera of patients with active TTP but not after their recovery, and that this activity was carried on PMP [75]. More recently, Galli et al. did a careful study of PMP in TTP, exhibiting the rise and fall of PMP with the course of disease [21] and suggest that PMP may be clinically relevant, ‘‘particlarly in microangiopathic forms’’ [21], a finding consistent with Ahn’s (that high PMP predominantly reflect small vessel thromboses, Section 5.2.3). Ahn et al. showed a similar rise and fall of circulating activated platelet aggregates (aPAg) in TTP, which correlated with clinical course, i.e. normalized in remission and rose in advance of relapses [246]. In view of Kelton’s findings cited above, this implies that aPAg rise and fall in parallel with PMP. 5.2.7.2. PMP in heparin-induced thrombocytopenia (HIT), other drug-induced thrombocytopenias. HIT is a drug induced immune thrombocytopenia in which antiheparin Ab and PF4 form an immune complex with heparin that binds to platelet FcR, causing platelet activation and destruction [365–368]. HIT Abs are potent platelet activators (as cited in [330]) and result in copious shedding of procoagulant PMP [369], leading those authors to propose that PMP account for the thrombotic complications in HIT-with-thrombosis (HITT) [369] and that PMP might serve as a diagnostic criterion of HITT [22]. Arnout has presented an hypothesis for the clinical manifestations of aPL based on parallels with HIT/ HITT, since the Abs of HIT also require a cofactor (PF4), and also have manifestations ranging from asymptomatic to severe thrombosis (HITT) [347]. Beyond the analogies given by Arnout are the facts that heparin is known to bind aPL antigen b2GPI [370], and that PF4 also binds to heparin, and has anti-coagulant activities similar to b2GPI, i.e. inhibition of prothrombinase, etc.

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[371] and cited in [372]. Thus Abs blocking either of these might contribute to thrombosis, as explicitly suggested by Chong et al. [330]. Ahn’s lab has confirmed high PMP in HIT/HITT, and has found that plasma from HITT patients is distinctly more potent in inducing activation of platelets and PAg in normal whole blood than that from HIT, and proposed this measure for discriminating HIT from HITT [373]. Boshkov et al. also recently attempted to discriminate HIT from HITT, finding that coagulation factors were depleted in both, indicating ‘‘global activation of the coagulation cascade’’, but no significant differences between HIT and HITT [374]. HIT Abs also activate cultured endothelial cells, including eliciting from them tissue factor (TF) [350], which in turn can activate platelets and coagulation; this action of HIT Abs was seen even without added heparin, but heparinase abolished this effect, suggesting that heparin-like molecules on the endothelial surface are involved, according to Arnout [347] in comments on [375]. Likewise, aCL/LAC IgG can cause activation of endothelial cells, also referenced in [347]. In trying to discriminate among various drug-induced thrombocytopenias, Warkentin et al. found that heparin induced, but not quinine/quinidine induced, thrombocytopenia showed elevated PMP bearing procoagulant activity, and suggested this as the explanation for the differing clinical syndromes; i.e. thrombotic tendency in the first, bleeding in the latter [376]. Their more recent publication on this topic was earlier commented on (see Section 5.2.6) [22].

5.2.8. PMP in neurologic disorders This article extends observations of PMP-associated CNS dysfunctions given above (see Sections 5.2 and 5.3) to topics in which platelet abnormalities are rarely considered in the current literature. It remains to be seen what role if any these abnormalities play in these conditions. 5.2.8.1. Multiple sclerosis (MS) and Alzheimer’s disease (AZ). Early literature described platelet abnormalities in patients with MS, specifically, increased adhesiveness [377,378], abnormal electrophoretic mobility [379,380], evidence of venous thrombosis at autopsy, easy bruising and thrombotic complications is reported in literature back to 1935; [381] and refs. cited therein. Mild thrombocytopenia and frequent elevation of platelet associated immunoglobulins were observed, suggesting immune-mediated platelet consumption in MS. In a more recent study, clear evidence of platelet activation in 33 MS patients was found, including elevated PMP, expresssion of activation marker CD62p, and plateletassociated IgM, all at P B0.001 [382]. In contrast, PMP assay of AZ patients revealed no elevation [12,383]. However, platelet activation marker

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CD62p and enhanced platelet–leukocyte interaction were found in AZ [383]. Abnormalities in platelet membrane fluidity have been documented in AZ by Zubenko et al. [384]. It has recently been shown that extracts of Gingko biloba are effective in retarding progression of AZ [385], relevant because major components of that botanical are potent inhibitors of PAF [386]. These observations suggest that some degree of platelet activation is common to both disorders (MS, AZ), though the mechanism and degree of activation apparently differs.

5.2.8.2. PMP in cardiopulmonary bypass (CPB) surgery, hemodialysis, etc.. Some 40–70% of patients undergoing CPB experience neurological complications manifesting as mild cognitive and somatic changes [387–391]. These effects resemble those cited above (see Section 5.2.2) in association with high PMP. George et al. reported elevated PMP in the setting of CPB [53], confirmed by Abrams et al. [20]. The PMP may arise in blood passage through the bypass equipment (see Section 4.6) since platelet activation has been reported in CPB since 1978, as referenced in [280,392,393], and see refs. 28–32 in [20]. George et al. reported a drop in GPs Ib and IIb on platelets following CPB [53], as has Wenger et al. [394], but Kestin et al., by a different method, could not confirm that [395]. It has been suggested that platelet secretory products could be neurotoxic [396], and efforts to inhibit those actions are reported [397], but preventing the ischemia in the first place is the ideal goal, perhaps by inducing a Scott-like platelet defect, as envisioned by Weiss and Lages [398]. 5.3. PMP in cell–cell interactions (see also Section 3.9) It is now appreciated that platelets and neutrophils (aka polymorphonucleocytes, PMN) are intimately linked in mutual interactions in processes of coagulation [121], inflammation, and hemostasis, either directly through cell–cell contact (adhesion) or indirectly via cytokines, tissue factor, etc. [119,399–401]. Sata et al. showed that LPS-stimulated monocyte MPs express tissue factor (TF) and prothrombinase activity, and preferentially bind thrombomodulin (TM) [124], a topic of continuing research, e.g. [402]. Cathepsin G, released by PMN, is a potent platelet activator [403]. With specific regard to PMP, their release is promoted by leukemic cells in culture [404], of interest in view of cancer-associated thromboses, and the same authors were earlier cited for showing release of MPs by leukocytes in phagocytosis [17]. Relatedly, it appears that PMP can be trapped in a thrombus, and evidence was earlier cited implicating a role for PMP in fibinolysis (see Section 4.2.3 and ref. [173]). While studying platelet–PMN interactions, Jy et al.

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noticed that the FITC-GP IIb/IIIa label of stimulated platelets found bound to PMN often had fluorescent intensity less than a single platelet, taken to indicate PMP bound to PMN [125]. Using two-color fluoresence microscopy, they showed that PMP bind to, activate, and link PMN together into grape-like clusters. Of related interest, Barry et al. have shown that PMP can act to enhance adhesion between monocytes and endothelial cells; and that PMP stimulate monocytes to upregulate CD11b/CD18 [126]. An often overlooked study showed that PMP promote platelet adhesion to the subendothelium of the rabbit aorta [405]. Incidentally, it was recently shown that the neutrophil GPs, CD11 and CD18, occur also on activated platelets [406].

6. Summary and concluding remarks The release of numerous PMP upon platelet activation is an efficent means for platelets to maximize their PPL surface for anchoring and assembling procoagulant or anticoagulant factors, thereby accelerating hemostasis locally at sites of activation. It is evident from this review that PMP almost certainly play additional roles in normal and pathological hemostasis. They participate in thrombus formation, fibrinolysis, leukocyte adhesion, platelet – endothelium interaction, and probably other activities, e.g. modulating phospholipase (see Section 3.5). This review covers the history of PMP, assay methodologies, factors influencing their production, their relevance in blood banking, but emphasizes studies of PMP in clinical disorders. A major purpose of this review is to bring these topics to wider attention in the hope that a consensus will emerge. Perhaps the most general controversy to be settled by future research is the extent to which PMP are causitive agents in pathology, or are mere epiphenomena, i.e. incidental consequences.

6.1. Clinical significance The clinical consequences of deficient PMP generation are well illustrated in Scott syndrome (see Section 5.1). Recent work that may bear on this includes new insights on the cytoskeleton [407], filopod formation [408] and prolonged bleeding in a rat model having defective filopods [409]. Conversely, the clinical relevance of elevated PMP is best illustrated in ITP (see Section 5.2.2). In this disorder, interaction of antiplatelet antibodies with platelets leads to PMP generation (see Section 4.4) but the degree of PMP elevation varies among patients. It was noted that ITP patients with high levels of PMP bleed little despite severe thrombocytopenia, and those with the highest levels of procoagulant PMP often suffer from small vessel TIA.

These observations imply that PMP in ITP are hemostatically active and in some cases thrombogenic [13]. Elevated PMP have been observed in many disorders in which platelet activation is documented. These include acute coronary syndromes (unstable angina) [318], small vessel strokes, multiple sclerosis [382], aPL Abs, ARDS and patients undergoing CPB [20,53] and probably dialysis [279] (see Section 4.6). High PMP are also seen in HIT, HITT, and TTP (see Section 5.2.7). Studies in limited numbers of patients showed that elevated PMP decreased with antiplatelet therapy such as aspirin or calcium channel blockers in ACS and small vessel TIA [12,316]. PMP normalized in parallel with recovery in TTP [21], in CPB and ARDS [20,53]. Although the cause of platelet activation differs among disorders, therapy for the underlying disease appears to normalize PMP by eliminating the stimulus for platelet activation. A more detailed discussion of potential therapies which specifically target membrane fragmentation is beyond the scope of this review. PMP assay appears useful in monitoring the efficacy of treatment.

6.2. PMP as marker of platelet acti6ation Regardless of controversies about their role, PMP may be viewed as a marker of platelet activation. It is increasingly appreciated that clinically practical assays for platelet activation are urgently needed in various thrombotic disorders [410]. PMP assay and CD62 are among the most promising because they are rapid and simple. PMP assay is particularly appealing because it is measured in platelet-poor plasma (PPP) and appear to be stable after separation of PPP, and if frozen for months. Other methods or markers of activation (e.g. CD62 expression, platelet aggregates, platelet–leukocyte association [411]) may supply more information but are subject to greater variability and must be performed within 4 h of blood drawing. We routinely measure the procoagulant activity of the PMP (functional assay) along with concentration determined by flow cytometry because PMP may differ in specific coagulant activity, and the flow cytometer does not dectect very small PMP, which in some cases may predominate [36]. Instrument set-up may require help of a technical specialist to maximize detection efficiency. An ELISA method was recently reported [27] as a promising alternative but its detection efficiency is difficult to evaluate in absence of independent standards. The key to a practical clinical PMP assay will rest upon inter-laboratory standards. Although a flow cytometer is an expensive instrument, many or most major clinical labs now have at least one for other applications such as lymphocyte phenotyping. We foresee clincal platelet assays rising to equal importance. PMP assay is simple and economical and may be feasible as a routine clinical test.

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Acknowledgements This work was supported by the Wallace H. Coulter Research Fund, the Charles Bosco Research Fund, the Mary Beth Weiss Research Fund, and gifts from Stanley Glaser and Kenneth N. Chasen. We are also grateful to the Beckman-Coulter Corporation for their continuing support.

Reviewers This article was kindly reviewed by Alan Schroit ([email protected]) and also by Shosaku Nomura, MD, First Department of Internal Medicine, Kansai Medical University, 10 – 15 Fumizono-cho, Moriguchi, Osaka 570, Japan.

Appendix A. Abbreviations ACL ACS AIDS AIHA ANA AnV aPC aPL APP ARDS AZ C CAD CAT CNS CPB CVA DAF DDAVP E EDRF FITC FL FLS GP GPI HIT HITT ITP LAC LS

anticardiolipin Ab acute cardiac syndrome acquired immune deficiency syndrome autoimmune hemolytic anemia antinuclear antibodies annexin V activated protein C (anticoagulant) antiphospholipid antibody amyloid precursor protein acute respiratory distress syndrome Alzheimer’s disease complement coronary artery disease computed axial tomography (scan) central nervous system cardiopulmonary bypass cerebrovascular accidents decay accelerating factor desmopressin erythrocytes; see also RBC endothelial relaxing factor (nitric oxide, NO) FL label, green: flourescein isothiocyanate fluoresence forward light scatter (flow cytometry) glycoprotein glycosyl phosphatidyl inositol (anchored proteins) heparin-induced thrombocytopenia HIT with thrombosis idiopathic (or immune) thrombocytopenic purpura lupus anticoagulant light scatter (in flow cytometry)

mAb MAC MI MP MRI MS PAF PAg PC PC PE PE PF3 PFP PGE1 Pi PMP PMN PNH PPL PRP PS RBC RVV RVVT SEM SIPA SLE SS TEM TIA TM TTP VEGF vWd vWf WBC

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monoclonal antibody membrane attack complex (of complement factors) myocardial infarct microparticles (Other Than Pmp) magnetic resonance imaging multiple sclerosis platelet activating factor platelet aggregates phosphatidyl choline; also: protein C (of the coagulation system) phosphatidyl ethanolamine; also: FL label red: phycoerythrin platelet factor 3 platelet-free plasma prostaglandin E1 inorganic phosphate platelet microparticles polymorphonucleocytes (neutrophils) paroxysmal nocturnal hemoglobinuria phospholipid platelet-rich plasma Phosphatidyl serine red blood cells; erythrocytes; also E Russell’s viper venom Russel’s viper venom time scanning electron microscope shear-induced platelet activation systemic lupus erythematosus side scattered light (flow cytometry) transmission electron microscope transient ischemic attacks thrombomodulin thrombotic thrombocytopenic purpura vascular endothelial growth factor von Willebrand disease von Willebrand factor white blood cells (leukocytes)

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Biographies Yeon S. Ahn joined the faculty of the University of Miami School of Medicine as Assistant Professor following his hematology fellowship in 1972, and became a Professor of Medicine in 1985. He is a graduate of the Seoul National University School of Medicine in Korea. His research and academic career developed under the mentorship of the late William J. Harrington. To-

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gether they pioneered new therapies for idiopathic thrombocytopenic purpura (ITP), introducing the use of vinca alkaloids (I.V., vinca-loaded platelets, slow infusions), colchicine, and danazol as immune modulators in ITP. His recent research focuses on platelet activation in ITP and thrombotic disorders in collaboration with Dr Wenche Jy and Lawrence Horstman. He is now director of the Wallace H. Coulter Platelet Laboratory at the Univesity of Miami, named in honor of his friend and supporter, the late Wallace H. Coulter, inventor of the Coulter principle for automatic blood cell counters. Lawrence L. Horstman received the B.S. degree in physical chemistry at Columbia University in 1977 and worked in Professor E. Racker’s mitochondrial research laboratory for 10 years, first at the Public Health Research Institute of the City of New York, then at Cornell University when Professor Racker moved there as Chairman of the Department of Biochemistry. Among his contributions to the study of oxidative phosphorylation was a method for removing the inhibitor from the ATPase called F1. Later studies focussed on respiratory control, submitochondrial particles, assessment of Mitchell’s chemiosmotic hypothesis, and purification of the Mg2 + -dependent ATPase inhibitor. More recently, he has been in Professor Yeon Ahn’s platelet research laboratory (Wallace H. Coulter Platelet Laboratory) since 1988. Aside from work related to platelet microparticles reported in this review, his work has focused on the alteration of platelet membranes and function in health and diseases, platelet fragmentation by complement, and the effects of lipophiles such as danazol on blood cell membranes.

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