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Originally published In Press as doi:10.1074/jbc.M403559200 on May 6, 2004

J. Biol. Chem., Vol. 279, Issue 29, 30697-30706, July 16, 2004
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Platelet Factor XIII and Calpain Negatively Regulate Integrin {alpha}IIb{beta}3 Adhesive Function and Thrombus Growth*

Suhasini Kulkarni and Shaun P. Jackson{ddagger}

From the Australian Centre for Blood Diseases, Department of Medicine, Monash Medical School, Box Hill Hospital, Box Hill, Victoria 3128, Australia

Received for publication, March 31, 2004 , and in revised form, April 16, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Excessive accumulation of platelets at sites of athero-sclerotic plaque rupture leads to the development of arterial thrombi, precipitating clinical events such as the acute coronary syndromes and ischemic stroke. The major platelet adhesion receptor glycoprotein (GP) IIb–IIIa (integrin {alpha}IIb{beta}3) plays a central role in this process by promoting platelet aggregation and thrombus formation. We demonstrate here a novel mechanism down-regulating integrin {alpha}IIb{beta}3 adhesive function, involving platelet factor XIII (FXIII) and calpain, which serves to limit platelet aggregate formation and thrombus growth. This mechanism principally occurs in collagen-adherent platelets and is induced by prolonged elevations in cytosolic calcium, leading to dramatic changes in platelet morphology (membrane contraction, fragmentation, and microvesiculation) and a specific reduction in integrin {alpha}IIb{beta}3 adhesive function. Adhesion receptor signal transduction plays a major role in the process by sustaining cytosolic calcium flux necessary for calpain and FXIII activation. Analysis of thrombus formation on a type I fibrillar collagen substrate revealed an important role for FXIII and calpain in limiting platelet recruitment into developing aggregates, thereby leading to reduced thrombus formation. These studies define a previously unidentified role for platelet FXIII and calpain in regulating integrin {alpha}IIb{beta}3 adhesive function. Moreover, they demonstrate the existence of an autoregulatory feedback mechanism that serves to limit excessive platelet accumulation on highly reactive thrombogenic surfaces.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The ability of platelets to adhere to the injured vessel wall and recruit other platelets is critical for hemostatic plug formation and vascular repair. Dysregulated platelet-platelet interactions leading to exaggerated thrombus growth contribute to the development of various cardiovascular disorders, including the acute coronary syndromes, thrombotic stroke, and the acute complications of peripheral vascular disease. Primary platelet adhesion to the damaged vessel wall is initiated by subendothelial matrix proteins, including collagen and von Willebrand factor (VWF),1 which engage specific receptors on the platelet surface, including glycoprotein (GP) Ib/V/IX, GPVI, and integrins {alpha}IIb{beta}3 and {alpha}2{beta}1 (1, 2). Firm adhesion of platelets is associated with granule secretion and extensive cytoskeletal remodeling, resulting in the development of a highly reactive surface for the recruitment of additional platelets. The reactivity of platelets is partly dependent on the surface immobilization of various adhesive ligands, including VWF, fibrinogen, and fibronectin, but is principally influenced by the activation status of integrin {alpha}IIb{beta}3 (1, 3, 4). The importance of this receptor in promoting hemostasis and thrombosis is well defined and therapeutic agents that target this receptor are important for the management of the acute coronary syndromes (5–8).

Given the central role played by integrin {alpha}IIb{beta}3 in supporting thrombus growth, considerable effort has been devoted to understanding the mechanism(s) regulating the adhesive function of this receptor. In particular, there has been intense interest in identifying the proximal signaling events regulating integrin {alpha}IIb{beta}3 activation. Central to this process is the mobilization of intracellular calcium, which is considered the principal initiator of integrin {alpha}IIb{beta}3 activation following platelet activation by a diverse range of stimuli (reviewed in Ref. 4). In fact, sustained calcium flux is not only important for initiating integrin {alpha}IIb{beta}3 activation (9, 10), but for maintaining this receptor in a high affinity state necessary for stable platelet aggregation (11). Cytosolic calcium flux in primary adherent and aggregating platelets can be maintained for long periods of time (up to 60 min),2 however, the significance of such prolonged calcium flux in terms of platelet adhesive function has not been well defined.

A major platelet enzyme that is preferentially activated by sustained levels of elevated calcium is the thiol protease, calpain. Activation of this protease is classically integrin {alpha}IIb{beta}3-dependent, occurs in the "late" phases of platelet stimulation and results in the limited proteolysis of a large number of platelet signaling and cytoskeletal proteins, many of which are physically or functionally linked to integrin {alpha}IIb{beta}3 (12). Moreover, calpain has been demonstrated to directly cleave the cytoplasmic tail of the {beta}3 subunit (13, 14), and based on studies in endothelial cells, such cleavage events may undermine the adhesive function of the receptor (15). The importance of calpain in regulating integrin {alpha}IIb{beta}3 adhesive function remains controversial with evidence that the protease may have both positive and negative effects on receptor function (16–18).

A second major platelet enzyme activated under conditions of sustained calcium flux is the cytosolic transglutaminase, factor XIII (FXIII). This enzyme is expressed at extremely high levels in platelets (at a concentration ~100-fold higher than plasma FXIII) where it has been demonstrated to covalently cross-link a broad range of cytoskeletal proteins, including actin, myosin, vinculin, and the small heat-shock protein, hsp27 (19). The fact that these are all proteins important for cytoskeletal remodeling have raised the possibility that cellular FXIII may participate in cytoskeletal reorganization in activated platelets. In contrast to the plasma forms of FXIII, which have a well established role in hemostasis through cross-linking fibrin monomers, there is limited information regarding a role for the cellular form of FXIII in platelet function, although recent studies by Dale et al. (20) suggest a role for this enzyme is serotonin-mediated localization of {alpha}-granular proteins on the activated platelet surface.

The studies detailed in this report describe for the first time an important functional role for platelet FXIII and calpain in negatively regulating the adhesive function of integrin {alpha}IIb{beta}3. We demonstrate that platelets undergoing a high level of prolonged cytosolic calcium flux exhibit FXIII and calpain-dependent morphological changes that are associated with a selective down-regulation in the adhesive function of integrin {alpha}IIb{beta}3. This reduction in integrin {alpha}IIb{beta}3 adhesiveness is most apparent on highly reactive adhesive substrates, such as type I fibrillar collagen and is associated with reduced platelet aggregate formation and thrombus growth. These findings demonstrate an important role for FXIII and calpain in limiting the excessive accumulation of platelets on potent thrombogenic surfaces.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
General Reagents and Antibodies—Fura Red-AM was from Molecular Probes Inc. Thapsigargin, ionophore A23187 [GenBank] , thimerosal, ADP, monodansylcadaverine (MDC), and p190–230 were from Sigma. Calpeptin, ALLN (N-acetyl-Leu-Leu-Nle-CHO), and E64-d were from Calbiochem Novabiochem. Antibodies against VWF (monoclonal antibody clone 5D2), P-selectin (polyclonal antibody), and GPIb{alpha} (monoclonal antibody clone WM23) were kind donations from Prof. Michael Berndt (Monash University, Australia). Antibodies against the intact heterodimer (monoclonal antibody clone P2) or active (monoclonal antibody clone PAC-1) forms of integrin {alpha}IIb{beta}3 were from Immunotech (France) and BD Biosciences, respectively. FITC-conjugated annexin V was from BD Biosciences. All other reagents were obtained from sources reported previously (21–23).

Preparation of Blood and Blood Cell Components—Blood and blood cell components were obtained from healthy donors or from an individual with FXIII deficiency (<1% plasma or platelet FXIIIa antigen), who had not taken any anti-platelet medication in the 2 weeks preceding the procedure. Blood was drawn into either 1:9 volume of trisodium citrate (15 mM) or hirudin (400 units/ml) for whole blood studies or theophyl-line containing acid/citrate/dextrose for platelet isolation. Washed platelets were prepared as described previously (22) and finally resuspended in modified Tyrode's buffer (10 mM Hepes, 12 mM NaHCO3, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 5 mM glucose) supplemented with 1 mM CaCl2/MgCl2 or 1 mM EGTA and 2 mM MgCl2 where indicated. Mouse platelets were isolated according to Goncalves et al. (24) and human red blood cells were prepared as previously described (25).

Static Adhesion Assays—Glass coverslips were left uncoated and unblocked (for adhesion to glass) or coated with 10 µg/ml VWF, 100 µg/ml or 2 mg/ml type I collagen, or 100 µg/ml fibrinogen overnight at 4 °C and then blocked with 5% human serum for 30 min. Washed platelets in Tyrode's buffer (1–3 x 107/ml) were allowed to adhere and spread for up to 60 min at 37 °C. Adherent platelets were fixed with 3.7% formaldehyde, or in 2% glutaraldehyde for scanning electron microscopy studies (23). In some experiments, platelets were pretreated with thapsigargin (10 nM) or thimerosal (1 µM) for 2 min prior to adhesion. In experiments examining the effect of calpain or FXIII inhibition on the rate of SCIP (sustained calcium-induced platelet morphology, see "Results") formation, platelets (1 x 107/ml) in Tyrode's buffer were pretreated with vehicle (Me2SO), 50 µg/ml calpeptin, 50 µM ALLN, 50 µM E64-d, 25 µM p190–230, or the indicated concentrations of MDC for 30 min, prior to being applied to glass or collagen. In experiments examining the role of platelet adhesion receptors and soluble agonists in SCIP formation, human platelets preincubated with vehicle or 40 µg/ml c7E3 Fab for 10 min, or murine wild type or FcR{gamma}-/-platelets, were allowed to adhere to VWF for 30 min or to collagen type I (100 µg/ml) for 15 min, respectively. Non-adherent platelets were aspirated and adherent platelets exposed to calcium-supplemented Tyrode's buffer alone, containing vehicle (Me2SO), 25 µM ADP, or 1 unit/ml thrombin, in the presence or absence of c7E3 Fab, for the indicated times. Adherent platelets were then fixed with 3.7% formaldehyde and visualized by differential interference contrast (DIC) microscopy (x63 lens, Leica DMIRB, Leica, Germany), images were captured with MCID software (Imaging Research Inc.) and scored as SCIPs if exhibiting a central membrane structure surrounded by smaller membrane bodies as seen in Fig. 1.



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  		FIG. 1.
Platelet morphological changes associated with a reduction in platelet surface reactivity. A, washed platelet monolayers were formed in the presence or absence of 1 mM CaCl2 for 30 min at 37 °C. DiOC6-labeled whole blood was then perfused over monolayers at 150 s-1 and the resulting platelet-monolayer interactions were monitored by fluorescence microscopy. The level of platelet adhesion to monolayers formed in the absence (-Ca) or presence (+Ca) of calcium after 2 min of perfusion is presented as mean ± S.E. (n = 3), * = p < 0.05. B, DIC images of platelets allowed to adhere to glass in the absence (-Ca) or presence (+Ca) of extracellular calcium. Note: arrowheads indicate fragmented platelets. Monolayers with lower than normal confluency are presented for optimal visualization of platelet morphology. Bar = 10 µm. C, a representative series of images of a single platelet undergoing SCIP formation on glass over the indicated time frame as visualized by real-time DIC video microscopy. Arrowheads indicate areas of fragmentation and microvesiculation. D, scanning electron microscopy images of platelets highlighting the specific sequence of morphological changes leading to SCIP conversion. Platelets rapidly convert from discs to spiny spheres to spread platelets within 2–5 min of adhesion after which the lamellae contract, numerous microvesicles are released, and the cell eventually fragments into a central membrane structure surrounded by smaller membrane bodies. Bar = 2 µm. E, images highlighting the process of membrane contraction and fragmentation.

 
Indirect Immunofluorescence—Washed platelets (1 x 107/ml) in Tyrode's buffer were applied to fibrinogen or VWF for 30 min at 37 °C in the presence of the appropriate primary antibody: anti-GPIb{alpha} (WM23, 2 µg/ml), anti-P-selectin (1 µg/ml), anti-fibrinogen (2 µg/ml), anti-VWF (5D2, 1 µg/ml), anti-intact integrin {alpha}IIb{beta}3 heterodimer (P2, 1 µg/ml), and anti-active integrin {alpha}IIb{beta}3 (PAC-1, 2 µg/ml). After 30 min, SCIP formation on fibrinogen was induced by adding 1 µM ionophore A23187 [GenBank] for 5 min. In experiments examining the effect of calpain or FXIII inhibitors on active integrin {alpha}IIb{beta}3 expression, platelets were pretreated with vehicle (Me2SO), ALLN (50 µM), MDC (25 µM), or MDC in conjunction with ALLN for 30 min prior to application to glass for 15 min. Adherent platelets were fixed, exposed to FITC-conjugated secondary antibody, and finally visualized using confocal-based DIC or fluorescence microscopy (x63 lens; Leica TCS-SP, Leica, Germany). To quantitate fluorescence associated with spread platelets or SCIPs, platelets were demarcated in DIC and the total fluorescence intensity and the surface area corresponding to those platelets quantitated using the Leica TCS-SP software quantitation module.

In Vitro Flow Assays—In studies examining thrombus growth, platelets in hirudin-anticoagulated whole blood were labeled with DiOC6 (1 µM) then perfused over a concentrated collagen matrix for 5 min at 1800 s-1 at 37 °C. In some studies, whole blood was incubated with vehicle (Me2SO), z-VAD-fmk (100 µM), calpeptin (50 µg/ml), ALLN (50 µM), MDC (25 µM), or a combination of calpeptin and MDC for 30 min prior to perfusion. In dose-response studies we found that the increase in thrombus growth observed with the indicated concentrations of inhibitors was not enhanced when up to 10-fold higher concentrations were used. Platelet adhesion and thrombus growth was monitored by confocal fluorescence microscopy and the thrombi were reconstructed and quantitated as previously described (23). In experiments correlating phosphatidylserine (PS) exposure (annexin V binding) with platelet aggregation, washed platelets (1.5 x 108/ml) loaded with 5 µM Fura Red-AM were reconstituted with packed red blood cells (50% hematocrit) and calcium-supplemented Tyrode's buffer containing 5 µg/ml FITC-annexin V. The platelet suspension was then perfused over collagen (2 mg/ml) at 1800 s-1 and the level of Fura Red and FITC-annexin V fluorescence was visualized by confocal microscopy (x63 lens; Leica TCS-SP). Under these conditions, non- or poorly activated platelets exhibited no annexin V but high Fura Red fluorescence (high intensity red), activated but non-SCIP platelets exhibited no annexin V but low-moderate Fura Red fluorescence (low intensity red), whereas SCIPs exhibited high annexin V but low Fura Red fluorescence (high intensity green).

In vitro flow-based aggregation assays examining the interaction of platelets in whole blood with immobilized platelet monolayers were performed according to Kulkarni et al. (23). Briefly, monolayers were formed by allowing washed platelets (2.5 x 108/ml) in Tyrode's buffer to spread in glass microcapillary tubes for 30 min at 37 °C. Citrated whole blood labeled with 1 µM DiOC6 was then perfused over the preformed monolayers at a shear rate of 150 s-1. Platelet-monolayer interactions were visualized by epifluorescence microscopy (x40 lens, Leica DMIRB, Leica, Germany) and video-recorded for off-line analysis as previously described (23).

Formation of SCIP Monolayers—Several independent approaches were investigated to achieve efficient (>95%) conversion of spread platelets to SCIPs in a preformed platelet monolayer. These include: 1) allowing platelets to form monolayers in the presence of extracellular calcium for prolonged periods of time (up to 120 min); 2) initially depleting platelet cytosolic calcium in a preformed monolayer with thapsigargin/EGTA for 15 min followed by exposure to thapsigargin/CaCl2 for a further 30 min; and 3) exposing preformed monolayers to 1 µM calcium ionophore A23187 [GenBank] in the presence of 1 mM CaCl2 for 5–10 min. Of all these strategies, we found that exposing platelet monolayers to ionophore A23187 [GenBank] was the most efficient method of inducing SCIP formation in ~100% of cells. The majority of the studies using a high percentage of SCIP monolayers were thus performed using ionophore A23187 [GenBank] as an inducer of SCIP formation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Calcium-induced Decrease in Platelet Surface Reactivity—We have previously established in vitro flow-based assays that enable real time analysis of cytosolic calcium flux during the development of platelet-platelet adhesion contacts (9, 25). Using these experimental systems we have established a critical role for intercellular calcium communication in promoting platelet aggregate formation and thrombus growth (11). We demonstrate here that prolonged exposure (up to 30 min) of preformed platelet monolayers to extracellular calcium (1 mM) induced a paradoxical decrease in surface reactivity toward other platelets (Fig. 1A, 34 ± 3.2% decrease, n = 3, p < 0.05). The inhibitory effects of extracellular calcium on platelet reactivity were time-dependent, in that platelet monolayers initially exposed to calcium were highly reactive, however. With time there was a progressive decline in platelet adhesiveness (data not shown). Morphological examination of monolayers revealed that all platelets adhering under calcium-free conditions exhibited the classic spread morphology, whereas up to 50% of calcium-exposed platelets adopted a distinct morphological form (Fig. 1B, arrowheads). These cells were typically characterized by a round central membrane structure (2–5 µm) surrounded by numerous smaller membrane fragments. For ease of reference, we will refer to these distinct morphological forms as SCIPs throughout the remainder of this report.

Real time analysis of platelet morphological changes revealed the temporal sequence of events leading to SCIP formation (Supplementary Materials Video 1). As shown in Fig. 1, C and D (upper panels), adherent platelets rapidly converted from discoid to spherical forms extending multiple filopodia followed rapidly by lamellipodial extension leading to a flattened, spread morphology within 2–5 min of adhesion. Platelets maintained this morphology for a variable period of time, ranging from 2 to 15 min. After this, fully spread platelets consistently underwent a specific sequence of morphological changes that was initiated by the marked contraction of lamellipodial membranes (Fig. 1, C and D, lower panels, and E), microvesiculation (Fig. 1C, arrows) and eventual fragmentation of the cell into a larger central membrane structure that was surrounded by smaller membrane bodies. Whereas the sequence of morphological changes from initial platelet adhesion to spreading typically took ~5 min, the conversion of fully spread platelets to the fragmented and vesiculating morphology was more rapid, occurring within a 1–3-min time frame once membrane contraction was initiated (Supplementary Materials Video 1).

SCIP Formation Occurs as a General Feature of Platelet Adhesion—To investigate the potential physiological significance of SCIP formation, platelet adhesion studies were performed on collagen, VWF, and fibrinogen matrices. We found that SCIP formation occurred on each of these adhesive matrices, although the proportion of platelets converting to SCIP morphology was much more prominent on a collagen surface relative to VWF (Fig. 2, A and B) and fibrinogen (data not shown). In fact, SCIP formation was a cardinal feature of platelet adhesion to fibrillar collagen, with ~75% of cells forming SCIPs within 10 min of initial adhesion (data not shown). Significantly, co-stimulation of VWF- (Fig. 2A) or fibrinogen-adherent (data not shown) platelets with soluble agonists such as ADP or thrombin dramatically enhanced the proportion of platelets converting to SCIPs, suggesting a synergistic role for soluble agonists and adhesive stimuli in this process. Similar morphological changes to those described here for SCIPs have previously been reported with collagen-adherent platelets (26) and have been demonstrated to be induced by sustained increases in cytosolic calcium. Consistent with this, we observed that ADP or thrombin stimulation of VWF-adherent platelets induced a high level of sustained cytosolic calcium flux in all platelets converting to SCIPs (data not shown). Similarly, co-stimulation of VWF-adherent platelets with reagents that directly potentiate cytosolic calcium flux, such as the sarcoendo-plasmic reticulum calcium ATPase inhibitor, thapsigargin, the inositol 1,4,5-trisphosphate receptor reductase, thimerosal, or ionophore A23187 [GenBank] , also potentiated SCIP formation (data not shown). High levels of cytosolic calcium flux induce PS expression on the surface of platelets leading to enhanced procoagulant function. Analysis of FITC-annexin V binding (a specific marker of PS expression) demonstrated a marked increase in PS exposure on the surface of SCIPs relative to resting and fully spread platelets (Fig. 2C). Pretreating platelets with EGTA, thereby preventing transmembrane calcium influx and sustained cytosolic calcium responses, eliminated PS exposure (data not shown) (27) and SCIP formation (Fig. 2B) on all adhesive surfaces examined. Taken together, the results in Figs. 1 and 2 raise the interesting possibility that high levels of sustained cytosolic calcium flux induce a phenotypic switch in platelets from a pro-adhesive to a procoagulant state.



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  		FIG. 2.
SCIP formation is a cardinal feature of platelet adhesion to collagen and is associated with PS exposure. A, washed platelets (1 x 107/ml) in Tyrode's buffer were allowed to adhere to either collagen (2 mg/ml) or VWF (10 µg/ml) for 30 min at 37 °C. VWF-adherent platelets were also exposed to 1 unit/ml thrombin (VWF + Thr) for 30 min prior to fixation to examine the effect of soluble agonist on VWF-induced SCIP formation. Adherent platelets were visualized by DIC and SCIP formation quantitated as described under "Experimental Procedures." Results are presented as mean ± S.E. (n = 3), ***, p < 0.01. B, scanning electron microscopy images of washed platelets allowed to adhere to collagen or VWF in the presence of 1 mM CaCl2 (Calcium) or 1 mM EGTA and 2 mM MgCl2 (EGTA/Mg) for the indicated times. Note: on both matrices, platelet fragmentation is calcium-dependent. These images are from one experiment representative of five. Bar = 2 µm. C, washed platelets were resuspended in calcium-supplemented buffer and allowed to spread on glass in the presence of FITC-annexin V (5 µg/ml) for 30 min. Platelets were imaged by DIC and confocal fluorescence microscopy (annexin V) to correlate platelet morphology with PS exposure. Note: only SCIPs bind significant levels of FITC-annexin V, whereas spread cells (some of which have been demarcated in white dotted lines in the annexin V panels) bind minimal levels of this marker. Bar = 10 µm.

 
Contribution of Adhesion Receptor Ligation to the Process of SCIP Formation—It is well established that platelet stimulation with physiological agonists alone induces minimal microvesiculation and PS exposure. We have confirmed these findings with respect to thrombin and ADP and have found that these agonists only induce significant microvesiculation and annexin V binding to the surface of primary adherent platelets (data not shown). To investigate the requirement for adhesion receptor signaling for membrane fragmentation and microvesiculation, we performed studies using murine platelets deficient in the FcR {gamma}-chain·GPVI receptor complex. These platelets are completely unresponsive to collagen stimulation, however, they respond normally to all other agonists (28). Consistent with previous reports, FcR{gamma}-/- platelets adhered to immobilized collagen but did not exhibit a cytosolic calcium response or undergo shape change (data not shown). Co-stimulation of these platelets with either ADP or thrombin elicited a robust cytosolic calcium response (data not shown) and induced platelet spreading, presumably through integrin {alpha}2{beta}1, however, there was minimal SCIP formation, even after prolonged periods of stimulation (Fig. 3A). This contrasted with wild type controls that exhibited similar morphological changes as observed in human platelets, with soluble agonist co-stimulation leading to an increase in the rate and extent of SCIP conversion (Fig. 3A). Similar to collagen, co-stimulation of VWF-adherent platelets with ADP or thrombin dramatically enhanced the rate of SCIP formation on human platelets (Fig. 3B). However, in the presence of the integrin {alpha}IIb{beta}3 blocking antibody, c7E3 Fab, neither agonist induced significant membrane fragmentation or microvesiculation (Fig. 3B). These studies suggest a major role for adhesion receptors in promoting SCIP formation in primary adherent platelets, presumably because of a requirement for adhesion receptor signal transduction in maintaining high levels of sustained cytosolic calcium flux.



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  		FIG. 3.
Contribution of adhesion receptor ligation to the process of SCIP formation. A, wild-type (WT) or FcR{gamma}-deficient (FcR{gamma}-KO) murine platelets (3 x 107/ml) were allowed to adhere to type I collagen (100 µg/ml) for 15 min. Non-adherent cells were aspirated and adherent cells were exposed to buffer alone (Control) or buffer containing 25 µM ADP or 1 unit/ml thrombin for the indicated times prior to fixation, visualization, and scored for SCIP formation. The images depict the morphology of either unstimulated wild type mouse platelets in the presence of calcium (WT-Ca) or in the presence of EGTA/MgCl2 (WT-EGTA) or FcR{gamma}-KO platelets in the presence of thrombin (FcR{gamma}-KO-Thr) at 30 min post-adhesion. The graphs depict the number of adherent platelets exhibiting SCIP morphology in WT or FcR{gamma}-KO platelets in the presence or absence of soluble agonist stimulation. The data are from a single experiment (5 fields) representative of three independent experiments. Bar = 5 µm. B, washed human platelets (2 x 107/ml) were incubated with vehicle (-c7E3) or 40 µg/ml of the integrin {alpha}IIb{beta}3 blocking antibody (+c7E3) for 10 min prior to being applied to VWF (10 µg/ml) for 30 min. Non-adherent cells were aspirated and adherent cells were exposed to buffer alone (Control), or buffer containing 25 µM ADP or 1 units/ml thrombin for the indicated times, in the presence of c7E3 where indicated. Platelets were imaged by DIC (upper panels) and the number of fragmenting and microvesiculating cells were quantitated. Images in the upper panels depict the morphology of -c7E3 platelets in the presence or absence of soluble agonist 30 min post-adhesion and are representative of three independent experiments. Data in C is presented as mean ± S.E. (n = 3); *, p < 0.05; **, p < 0.01; ***, p < 0.001. Bar = 10 µM.

 
SCIP Formation Is Partially Regulated by Calpain—The calcium-dependent thiol protease, calpain, has a well defined role in promoting membrane microvesiculation from the surface of platelets (29, 30). To investigate the role of this protease in regulating the morphological features relevant to SCIP formation, platelets were pretreated with the calpain inhibitors, ALLN, calpeptin, or E64-d prior to performing adhesion studies on glass or collagen. As demonstrated in Fig. 4, calpain inhibitors did not inhibit the ability of platelets to spread or contract their lamellar membranes post-spreading (Fig. 4A, arrows), however, they significantly decreased the proportion of platelets undergoing membrane fragmentation and vesiculation at all time points examined (Fig. 4, A and B). Similarly, both inhibitors dramatically reduced SCIP formation when platelets adhered to collagen type I fibrils (data not shown). This decrease in SCIP formation was unlikely to be because of a general decrease in the rate of platelet activation as calpeptin and ALLN-treated platelets adhered and spread at a similar rate to vehicle-treated platelets and their mean and peak calcium levels were not significantly reduced compared with control platelets (data not shown). Pretreating platelets with the cell-impermeable calpain inhibitor, leupeptin, had no effect on SCIP formation (data not shown) indicating that intracellular calpain is primarily responsible for mediating the morphological conversion of spread platelets to SCIPs. In control studies, we confirmed that the effects of the membrane-permeable calpain inhibitors were specific, as inhibitors against other thiol proteases, such as cathepsins (CA-074Me), had no effect on SCIP formation (data not shown).



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  		FIG. 4.
Calpain and platelet FXIII cooperatively regulate SCIP formation. A–D, washed platelets (1 x 107/ml) resuspended in calcium-supplemented Tyrode's buffer were incubated with vehicle (Control), 50 µg/ml calpeptin (CP) alone, 25 µM MDC alone (unless otherwise indicated as in C), or in combination (MDC + CP) for 30 min prior to application to glass (A–C) or type I collagen (D) for up to 30 min at 37 °C. Platelets were then visualized by DIC microscopy (A) and the level of SCIP formation was quantitated as described under "Experimental Procedures" (B–D). Images in A are of platelets adhering to glass at 15 min and are representative of four independent experiments. E and F, platelets from a Normal donor or an individual with FXIII deficiency (<1% platelet FXIII) (FXIII-d) were pretreated with vehicle or 50 µg/ml calpeptin (FXIII-d + CP), then applied to collagen (E) or glass (F) for up to 30 min prior to fixation, visualization, and scoring for SCIP formation. Data in E depicts mean levels of SCIP formation under conditions described using FXIII-d platelets from a single donor in 10 random fields. Representative images from this experiment are presented in F. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Bar = 10 µm.

 
Role of Platelet Factor XIII in Promoting SCIP Formation— Our studies indicate an important, albeit partial role for calpain in regulating the conversion of spread platelets to SCIPs. Given the complete abrogation of this process in the presence of EGTA, other calcium-dependent enzymes are likely to be involved. Another well defined calcium-dependent enzyme expressed at very high levels in platelets is the blood coagulation transglutaminase, FXIII. Approximately 50% of total FXIII activity is stored in platelets, however, the role of this transglutaminase in platelet function remains unclear (19). Similar to calpain, platelet FXIII requires high levels of cytosolic calcium to induce enzyme activation (19), suggesting that it may be a potential candidate for inducing SCIP formation. To investigate this possibility, platelets were pretreated with the competitive substrate of transglutaminases, MDC. MDC dose dependently inhibited SCIP formation on glass (Fig. 4C) and all other substrates examined (data not shown). Inhibition of SCIPs was not because of indirect effects of MDC on platelet activation or calcium flux as there was no significant difference in the ability of platelets to spread (Fig. 4A) or exhibit a sustained oscillatory calcium response (data not shown). Similar to calpain inhibitors, the effect of MDC on SCIP formation was partial (Fig. 4, A, C, and D), however, concurrent inhibition of both calpain and transglutaminases inhibited SCIP formation by >95% on all adhesive surfaces examined (Fig. 4, A and D). Significantly, pretreating platelets with a membrane-impermeable peptide inhibitor of FXIIIa (p190–230) markedly attenuated SCIP formation, implicating a major role for the extracellular form of this enzyme in promoting SCIP formation (data not shown).

MDC is a competitive transglutaminase substrate and does not differentiate between FXIII and other transglutaminases. To investigate the relative importance of FXIII versus other transglutaminases in SCIP formation, studies were performed with platelets congenitally deficient in FXIII (<1% plasma and platelet FXIII). Similar to MDC-treated platelets, platelets lacking FXIII exhibited a marked defect in SCIP formation (Fig. 4E). Furthermore, pretreatment of FXIII-deficient platelets with calpain inhibitors inhibited SCIP formation by >90% at all time points examined (Fig. 4, E and F). These findings support a major role for platelet FXIII in promoting the conversion of platelets to SCIPs.

SCIP Formation Leads to a Down-regulation in the Adhesive Function of Integrin {alpha}IIb{beta}3—To gain insight into the molecular mechanism(s) by which SCIP conversion leads to a decrease in platelet surface reactivity, the dynamics of platelet adhesion on SCIP monolayers were examined. In all studies, we observed a strong correlation between the proportion of platelets exhibiting morphological characteristics of SCIPs and the efficiency with which these monolayers supported platelet-platelet adhesive interactions. For example, when monolayers contained ~40–50% SCIPs, there was a corresponding ~30% reduction in the number of platelets forming stable adhesive contacts (see Fig. 1A). When >95% of platelets in a monolayer were converted to SCIPs (by artificially increasing the cytosolic calcium level in all cells, see "Experimental Procedures"), there was a ~80% decrease in adhesion (Fig. 5A). The low level of adhesion on SCIP monolayers was not because of a reduced ability of these cells to recruit platelets from bulk flow (Fig. 5B) but rather a reduced capacity of these cells to support stable adhesion of tethered platelets (Fig. 5C). For example, whereas 28.7 ± 9.4% (n = 3) of tethered platelets formed firm adhesion contacts on spread platelet monolayers within 30 s of perfusion, only 3.8 ± 2.2% (n = 3) of tethered platelets formed stable platelet-platelet interactions on SCIP monolayers at the same time point (Fig. 5C). Inhibition of SCIP formation by pretreating platelets with calpeptin and MDC prevented the calcium-induced decrease in platelet reactivity (data not shown), providing further evidence that SCIP conversion plays a major role in regulating platelet-platelet adhesive interactions.



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  		FIG. 5.
SCIP monolayers exhibit decreased capacity to support firm platelet-platelet adhesion contacts under flow. Spread platelet or SCIP monolayers were formed as described under "Experimental Procedures." Whole blood incubated with DiOC6 was perfused over spread platelet or SCIP monolayers at 150 s-1 and the resulting platelet-monolayer interactions were monitored by fluorescence microscopy and quantitated off-line. A, these results demonstrate the level of platelet adhesion on monolayers at 5, 30, and 60 s of perfusion. The data in B depicts the level of platelet tethering in the first 5 s of perfusion and in C shows the proportion of tethering platelets forming stationary adhesion contacts at the indicated times during perfusion. All results are presented as mean ± S.E. (n = 3). **, p < 0.01.

 
Given the importance of integrin {alpha}IIb{beta}3 in sustaining platelet-platelet adhesive interactions during aggregate formation (5–7), we examined the possibility that conversion of platelets to SCIPs led to a down-regulation in the adhesive function of integrin {alpha}IIb{beta}3. As shown in Fig. 6A, whereas intact spread platelets expressed high levels of active integrin {alpha}IIb{beta}3 (monitored using the activation-dependent antibody, PAC-1), platelets that had undergone conversion to SCIPs exhibited ~75% (n = 3, p < 0.01) reduction in PAC-1 binding (Fig. 6, A and B; Table I). This decrease in PAC-1 binding was observed on the surface of all SCIPs, regardless of the experimental conditions or substrates utilized to induce SCIP formation (data not shown). Furthermore, a similar reduction in the binding of fluorescently labeled fibrinogen was observed under the same experimental conditions (data not shown). In contrast, the overall level of intact integrin {alpha}IIb{beta}3 heterodimer (representing the sum of resting and activated receptor), as assessed by the complex-dependent anti-integrin {alpha}IIb{beta}3 antibody P2, was not significantly different between spread platelets or SCIPs (Fig. 6C; Table I). Furthermore, the surface expression of other proteins involved in promoting platelet-platelet interactions, including VWF, GPIb, P-selectin, and fibrinogen were not significantly different between the two cell morphologies (Table I). These findings suggest that conversion to SCIPs leads to a selective down-regulation in the adhesive function of integrin {alpha}IIb{beta}3.



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  		FIG. 6.
SCIPs exhibit a selective decrease in the surface expression of active integrin {alpha}IIb{beta}3. A–C, washed platelets (1 x 107/ml) were applied to fibrinogen-coated coverslips for 30 min at 37 °C in the presence of antibodies against the active (A and B) or intact (C) conformation of integrin {alpha}IIb{beta}3. Note: a high proportion of platelets in A and B were converted to SCIPs by treatment with 1 µM ionophore A23187 [GenBank] for 5 min prior to fixation. D, vehicle (Control), 50 µM ALLN, 25 µM MDC, or MDC + ALLN-treated platelets were applied to glass in the presence of anti-active {alpha}IIb{beta}3 antibody for 15 min prior to fixation. In all studies, the morphology of adherent platelets was visualized by DIC microscopy, whereas the surface expression of intact (Intact {alpha}IIb{beta}3) and active (Active {alpha}IIb{beta}3) integrin was visualized by confocal microscopy. Quantitation of fluorescence intensity was performed as described under "Experimental Procedures." Note: arrowheads in A and C highlight spread cells that have converted to SCIPs. The images are from one experiment representative of six and the data in B and D are presented as mean ± S.E. (n = 3); *, p < 0.05; ***, p < 0.001. Bar = 10 µm.

 


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  		TABLE I
Surface expression of membrane proteins on spread platelets versus SCIPs

 
Calpain and FXIII Inhibitors Sustain Integrin {alpha}IIb{beta}3 Activation—To examine more directly the role of calpain and platelet FXIII in regulating the ligand binding function of integrin {alpha}IIb{beta}3, platelets were pretreated with calpain and/or FXIII inhibitors and the level of PAC-1 binding was quantitated. As demonstrated in Fig. 6D, we observed a strong inverse relationship between the proportion of platelets exhibiting SCIP morphology and their ability to bind PAC-1. Pretreating platelets with calpain or FXIII inhibitors reduced the proportion of SCIPs, leading to a corresponding increase in the surface expression of active integrin {alpha}IIb{beta}3 (Fig. 6D). We consistently noted that platelets beginning to undergo transformation to SCIPs, as evidenced by contraction of the lamellar membranes, exhibited a similar decrease in PAC-1 binding to that observed in platelets that had completed the transition to the SCIP morphology. Furthermore, pretreatment with calpain or FXIII inhibitors did not delay the loss in PAC-1 binding once SCIP formation had been initiated, indicating that the down-regulation of integrin {alpha}IIb{beta}3 activity occurs early in the process of SCIP formation. Inhibiting both enzymes concurrently prevented both platelet fragmentation and the loss of integrin {alpha}IIb{beta}3 in >80% of adherent platelets (Fig. 6D). Collectively, these results demonstrate an important role for calpain and platelet FXIII in down-regulating the adhesive function of integrin {alpha}IIb{beta}3.

Calpain and Platelet FXIII Negatively Regulate Platelet Aggregate Formation and Thrombus Growth—To investigate the potential physiological significance of calpain and FXIII-mediated down-regulation of integrin {alpha}IIb{beta}3 adhesive function, the effect of calpain inhibitors and/or MDC on platelet thrombus formation was investigated on a type I fibrillar collagen matrix. For these studies, anticoagulated whole blood was perfused through collagen-coated microcapillary tubes and the rate and extent of thrombus growth was examined by confocal microscopy. As demonstrated in Fig. 7, A and B, both the rate and extent of thrombus growth was significantly enhanced by pretreating platelets with either calpain or FXIII inhibitors alone. Concurrent inhibition of both enzymes resulted in an additive effect on thrombus growth, resulting in an approximate doubling in total thrombus mass at each of the time points examined (p < 0.001) (Fig. 7, A–C). This increase in thrombus volume resulted from both an increase in the number of smaller thrombi (Fig. 7C, oblique view) as well as size of individual thrombi (Fig. 7C, side view). In control studies, we confirmed that the increase in thrombus size induced by MDC and calpeptin was not because of a nonspecific increase in platelet reactivity, as these inhibitors did not enhance platelet aggregation induced by threshold concentrations of ADP and thrombin receptor agonist peptide (data not shown). Furthermore, these inhibitors had no enhancing effect on thrombus growth on a VWF matrix, a finding consistent with the inability of this matrix to induce significant SCIP formation under these experimental conditions (data not shown).



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  		FIG. 7.
Inhibition of SCIP formation leads to accelerated thrombus growth on collagen. A–C, DiOC6-labeled treated blood was pretreated with vehicle (Control), 50 µg/ml calpeptin (CP), 25 µM MDC or calpeptin, and MDC (MDC + CP) for 30 min prior to perfusion over collagen (2 mg/ml) for 5 min. Thrombus growth was monitored by real-time confocal microscopy and quantitated. A, data are from one experiment representative of six. B, thrombus growth after 5 min of perfusion. Data are presented as mean ± S.E. (n = 6). *, p < 0.05; ***, p < 0.001. C, reconstructed images of thrombi formed by either vehicle (Control)orMDC + CP-treated platelets in whole blood at 5 min of perfusion on collagen. Note: upper panels, oblique view; lower panels, side perspective. Images are from one experiment representative of five. D and E, Fura Red-labeled platelets (1.5 x 108/ml) were reconstituted with erythrocytes (50% hematocrit) as described under "Experimental Procedures" and perfused over collagen at 1800 s-1 in the presence of FITC-annexin V and visualized by confocal microscopy. Note: black-green look-up table, annexin V fluorescence; black-yellow look-up table, Fura Red fluorescence. Image in D depicts a field at 5 min of perfusion. Bar = 10 µm. E, time series of Fura Red-low cells supporting stable platelet-platelet interactions (upper panels) and annexin V-positive cells failing to support stable platelet-platelet adhesion (lower panels). These images are from one experiment representative of five separate perfusion experiments. Bar = 2 µm.

 
To examine more directly the effect of SCIP formation on platelet aggregation we performed perfusion studies on platelets dual-labeled with the calcium indicator dye, Fura Red, and FITC-annexin V. Fluorescence emission from Fura Red decreases with increased cytosolic calcium, whereas FITC-annexin V only binds to the surface of SCIPs, thus an increased FITC-annexin V/Fura Red ratio is indicative of SCIP transformation. Annexin V positive cells appeared as early as 30 s during perfusion of whole blood over a collagen substrate indicating that the formation of SCIPs is a relatively early event in the thrombotic process. In addition, the majority (>95%) of annexin V binding occurred on the surface of primary adherent platelets (Fig. 7D). More detailed examination of platelet-platelet interactions revealed that only platelets exhibiting minimal FITC-annexin V fluorescence supported the stable adhesion of tethering platelets and became nuclei for platelet aggregation (Fig. 7E, upper panels; Supplementary Materials Video 2). Those platelets displaying a high ratio of annexin V:Fura Red fluorescence (indicative of SCIPs) consistently failed to support stable platelet-platelet interactions and never supported thrombus development (Fig. 7E, lower panels; Supplementary Materials Video 3). Taken together, these studies define an important role for SCIP transformation in negatively regulating platelet aggregation and thrombus formation on a collagen substrate.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The results presented here demonstrate for the first time a novel autoregulatory feedback mechanism, linked to sustained cytosolic calcium flux, which serves to dampen platelet adhesive function and limit thrombus growth. This calcium-dependent signaling mechanism involves activation of platelet FXIII and calpain, leading to a selective down-regulation in the adhesive function of integrin {alpha}IIb{beta}3. This mechanism appears to primarily operate in platelets activated by potent thrombogenic stimuli and thus, may have evolved to limit excessive platelet accumulation on highly reactive adhesive substrates. The demonstration that high, sustained levels of cytosolic calcium coordinately regulate microvesiculation, PS exposure, and integrin {alpha}IIb{beta}3 adhesive function suggests a key role for platelet FXIII and calpain in regulating the phenotypic switch of platelets from a proadhesive to a procoagulant state.

Our studies have defined for the first time an important functional role for platelet FXIII in regulating integrin {alpha}IIb{beta}3 adhesive function. Moreover, they have demonstrated that surface expression of this enzyme is important for its ability to down-regulate platelet surface reactivity. Whereas the mechanism for FXIII expression on the surface of platelets has not been established, it is of interest that the major binding site for the plasma form of FXIII has been identified as integrin {alpha}IIb{beta}3 (31). FXIII covalently cross-links glutamyl and lysine residues on a broad range of protein molecules, including several key adhesive proteins such as fibronectin, thrombospondin, fibrinogen, and VWF (19, 20). Thus, FXIII-mediated covalent cross-linking of one or more integrin {alpha}IIb{beta}3 ligands, and possibly the integrin {alpha}IIb{beta}3 itself (32), may contribute to the ability of this enzyme to down-regulate platelet adhesive function. The mechanism by which surface FXIII regulates membrane fragmentation and microvesiculation is more difficult to explain, but is presumably related to secondary effects of this enzyme on adhesion receptor signal transduction necessary for the induction of these platelet responses. Several other important issues that will require further investigation include examination of the potential contribution of the plasma form of FXIII in regulating integrin {alpha}IIb{beta}3 adhesive function, as well as identifying the functional importance of the FXIII-platelet interactions in terms of modulating the severity of the bleeding tendency associated with FXIII deficiency.

The mechanisms utilized by calpain to regulate integrin {alpha}IIb{beta}3 adhesive function remain unclear, although a number of possibilities can be envisaged. One possible mechanism that may induce integrin {alpha}IIb{beta}3 down-regulation involves calpain cleavage of the {beta}3 cytoplasmic domain of {alpha}IIb{beta}3. Studies by Du et al. (13) have shown that calpain can cleave the cytoplasmic domain of the {beta}3 subunit in ionophore A23187 [GenBank] -stimulated platelets, a process that may induce conformational changes in the extracellular domains of the integrin leading to receptor inactivation. Consistent with such a possibility are studies in apoptotic endothelial cells, in which cleavage of the cytoplasmic tail of integrin {alpha}v{beta}3 was associated with reduced integrin-mediated cell adhesiveness (15). An alternative mechanism relates to calpain-mediated cleavage of various signaling enzymes (FAK, Src, and PTP-1B) and cytoskeletal proteins (talin, filamin-1, and cortactin) (33–37). Several of these proteins have been implicated in the regulation of integrin {alpha}IIb{beta}3 adhesive function (38, 39) and cleavage and disassembly of cytoskeletal signaling complexes may undermine the sustained activation of integrin {alpha}IIb{beta}3 and/or its cytoskeletal anchorage (16). These mechanisms are not necessarily mutually exclusive and it remains possible that each may contribute in varying degrees to the down-regulation of integrin {alpha}IIb{beta}3 adhesive function.

We have demonstrated that the conversion of spread platelets to SCIPs is a general feature of platelet adhesion to physiological substrates, although the efficiency of the conversion was much greater on fibrillar collagen relative to VWF or fibrinogen. This difference can be explained by the distinct calcium kinetics induced by these different substrates, with collagen inducing high, sustained oscillatory calcium flux, whereas VWF and fibrinogen typically inducing a lower, oscillatory calcium response (9, 11). In this context, it is of interest that this difference in calcium signaling underpins the initial difference in platelet surface reactivity between collagen and VWF, explaining the relative thrombogenic potential of these substrates (11). Our findings raise the interesting possibility that this calcium-induced increase in platelet surface reactivity may be self-limiting, due in part, to calcium-dependent calpain and FXIII activity. Thus calcium appears to play a dual role in regulating platelet reactivity, initially by promoting integrin {alpha}IIb{beta}3 activation, then at later time points, by stimulating calpain- and FXIII-induced down-regulation of integrin {alpha}IIb{beta}3.

The demonstration that calcium-induced FXIII and calpain activation coordinately regulates membrane microvesiculation, PS exposure, and integrin {alpha}IIb{beta}3 down-regulation, suggests a key role for these enzymes in regulating the phenotypic conversion of platelets from a proadhesive to a procoagulant state. Moreover, the demonstration that these changes primarily occur in adherent platelets provides a likely explanation as to why previous studies examining soluble agonist stimulation of platelets in suspension have failed to identify this important functional linkage. The requirement for co-stimulation between adhesion receptors and soluble agonists for efficient SCIP formation presumably reflects the synergy between these receptors in terms of maintaining high levels of sustained cytosolic calcium flux, necessary for FXIII and calpain activation. It is also possible that spatial differences in adhesion receptor signaling potentially linked to submembranous calcium flux are required for efficient calpain and FXIII activation, a possibility that will require further investigation.

Platelet morphological changes relevant to SCIPs have previously been demonstrated in vivo, primarily in platelets juxtaposed to collagen fibrils at sites of vascular injury (40–44). Our findings that SCIP conversion leads to down-regulation of platelet surface adhesiveness may provide a partial explanation for previous observations that platelets in direct contact with exposed subendothelium eventually become non-reactive toward other platelets (40, 45, 46). Given the central role played by integrin {alpha}IIbb3 in hemostasis and thrombogenesis, unraveling the molecular mechanisms utilized by FXIII and calpain to regulate integrin {alpha}IIbb3 receptor function may have pathophysiological importance. Ultimately, such information may help identify individuals at risk of thrombosis because of defects in this integrin {alpha}IIbb3 regulatory mechanism, and perhaps in the longer term, may uncover new approaches to regulate platelet reactivity in vivo.


   FOOTNOTES
 
* This work was supported by the National Health and Medical Research Council, The National Heart Foundation, and The Wellcome Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

The on-line version of this article (available at http://www.jbc.org) contains Videos 1-3. Back

{ddagger} To whom correspondence should be addressed: Australian Centre for Blood Diseases, Dept. of Medicine, Monash University, Level 5 Clive Ward Bldg., Arnold St., Box Hill, Victoria 3128, Australia. Tel.: 61-3-9895-0350; Fax: 61-3-9895-0332; E-mail: shaun.jackson{at}med.monash.edu.au.

1 The abbreviations used are: VWF, von Willebrand factor; MDC, monodansylcadaverine; SCIP, sustained calcium-induced platelet morphology; PS, phosphatidylserine; ALLN, N-acetyl-Leu-Leu-Nle-CHO; FITC, fluorescein isothiocyanate; GP, glycoprotein; DIC, differential interference contrast. Back

2 S. Kulkarni and S. P. Jackson, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank Hilary Hoare for technical assistance and Warwick Nesbitt, Hatem Salem, and Varuni Kanagasundaram for helpful discussions and constructive comments throughout this project. Thanks also to the generous donations of blood from our donors and to Sascha Hughan for providing the DIC time-lapse video.



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S. Kulkarni, K. J. Woollard, S. Thomas, D. Oxley, and S. P. Jackson
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S. M. Jobe, L. Leo, J. S. Eastvold, G. Dickneite, T. L. Ratliff, S. R. Lentz, and J. Di Paola
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G. Remenyi, R. Szasz, P. Friese, and G. L. Dale
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