SEMINARS IN THROMBOSIS AND HEMOSTASIS—VOLUME 18, NO. 1, 1992

Thrombin-Induced Down-Regulation of the Platelet Membrane Glycoprotein Ib-IX Complex

Thrombin is a potent physiologic stimulus for plate­ let aggregation.1-3 Thrombin modulates the platelet sur­ face expression of receptors for adhesive glycoproteins involved in platelet adhesion and aggregation. Thus, thrombin up-regulates the platelet surface expression of the fibrinogen,4 von Willebrand factor,5 fibronectin,6 and vitronectin7 receptors on the glycoprotein (GP) IIb/ IIIa complex. In addition, thrombin up-regulates the platelet surface expression of GMP-140 (reflecting α granule secretion)8 and both 110 kd (LAMP-1) and 53 kd lysosomal granule proteins (reflecting lysosomal secre­ tion). 9,10 In striking contrast, thrombin down-regulates the platelet surface expression of the von Willebrand factor receptor on the GPIb-IX complex. 11-15 This article will review the current state of knowledge with regard to this thrombin-induced down-regulation of the platelet membrane GP Ib-X complex.

STRUCTURE OF THE GLYCOPROTEIN Ib-IX COMPLEX GPIb and GPIX are two distinct integral membrane proteins that form a noncovalent equimolar complex in the platelet membrane.16,17 GPIb and GPIX have appar­ ent molecular masses of approximately 170 and 18 kd, respectively.16 GPIb is a disulfide-linked two-chain mol­ ecule consisting of an α chain and a β chain, with appar­ ent molecular masses of approximately 140 and 24 kd, respectively.16 GPIX is a single chain polypeptide. GPIba, GPIbp, and GPIX are all members of the leu-

From the Department of Pediatrics, University of Massachusetts Medical School, Worcester, Massachusetts. Reprint requests: Dr. Michelson, Associate Professor of Pediat­ rics, Pathology, and Immunology, Department of Pediatrics, Univer­ sity of Massachusetts Medical School, 55 Lake Avenue North, Worces­ ter, MA 01655. 18

cine-rich α2-glycoprotein (LRG) family.18 There are ap­ proximately 25,000 copies of both GPIb and GPIX ex­ posed on the surface of each resting platelet.17,19 Only the GPIIb/IIIa complex, with approximately 50,000 cop­ ies on the surface of each platelet,20 is more abundant. Platelet surface GPIb is directly linked to the platelet cytoskeleton via actin-binding protein.21,22 In addition to the platelet surface pool of GPIb, evidence has been provided for both intraplatelet14,23-25 and plasma pools.26 The intraplatelet pool of GPIb may be located in the open surface canalicular system.14 The plasma pool is in the form of glycocalicin, a soluble proteolytic frag­ ment of GPIba that is generated by cleavage in a position close to the transmembrane domain of the protein.26,27

FUNCTION OF THE GLYCOPROTEIN Ib-IX COMPLEX GPIb is a receptor for both von Willebrand fac­ tor16,28 and thrombin.16,29 The amino terminal domain of GPIba contains a binding site for von Willebrand factor that is involved in platelet adhesion to damaged blood vessel walls (reviewed by Ruggeri16). In addition, the amino-terminal domain of GPIba contains a binding site for thrombin that is involved in platelet activation (re­ viewed in Ruggeri16 and Jamieson29). However, there are GPIb-dependent and GPIb-independent pathways of thrombin-induced platelet activation.30 Thus, a thrombin receptor unrelated to GPIb has recently been identified.31 The functions of GPIbβ and GPIX remain unknown, although phosphorylation of GPIbβ may play a role in the regulation of actin polymerization.32

DOWN-REGULATION OF THE PLATELET SURFACE GLYCOPROTEIN Ib-IX COMPLEX George et al11 were the first to report that thrombin stimulation results in decreased binding to washed plate-

Copyright © 1992 by Thieme Medical Publishers, Inc., 381 Park Avenue South, New York, NY 10016. All rights reserved.

Downloaded by: NYU. Copyrighted material.

ALAN D. MICHELSON, M.D.

DOWN-REGULATION OF GLYCOPROTEIN COMPLEX—MICHELSON

19

lets of a monoclonal antibody directed at the von Willebrand factor binding site on GPIb. We 12,15,30 and oth­ ers 13,14 have further investigated this phenomenon. From these studies 11-15,30 the following conclusions can be drawn.

In order to determine whether thrombin could in­ duce down-regulation of the GPIb/IX complex in the physiologic setting of whole blood, we modified a previ­ ously described33 flow cytometric method in two essen­ tial ways.15 First, to prevent thrombin-induced fibrin clot formation, the peptide glycyl-L-prolyl-L-arginyl-L-proline (GPRP) (Calbiochem, San Diego, CA) was included in the assay to inhibit fibrin polymerization.34-36 GPRP is stable, resistant to proteolytic agents (including throm­ bin), and does not suppress thrombin activity.34 Second, to enable the detection of activation-dependent changes in the GPIb/IX complex, a GPIV-specific monoclonal antibody (OKM5), rather than a GPIb-specific mono­ clonal antibody,33 was used to identify platelets in whole blood.15 Monocytes, the only other circulating hemopoi­ etic cells to which OKM5 binds,37 were excluded from analysis by gating on light scatter. In this whole blood system, purified human α-thrombin induced marked reductions in the platelet surface binding of monoclonal antibodies 6D1 (directed against the von Willebrand factor binding site on GPIb), FMC25 (directed against GPIX), and AK1 (directed against the GPIb/IX complex) (Fig. 1A). The maximal thrombin-induced decreases in binding were 79.2 ± 2.1% (6D1), 75.1 ± 4.2% (FMC25), and 60.0 ± 4.2% (AK1) (mean ± SEM, n = 3). The possibility that the thrombin-induced decrease in binding of GPIb-specific antibodies is due to occupation of the antibody binding site by thrombin bound to its receptor on GPIb38,39 is excluded by the thrombin-induced decrease in the platelet surface binding of multiple monoclonal antibodies di­ rected against different epitopes12,14,15,30 and the fact that these antibodies do not interfere with thrombin bind­ ing to platelets.38 In contrast to the findings with the GPIb/IX com­ plex, thrombin resulted in marked increases in the plate­ let surface binding of monoclonal antibodies S12 (GMP140-specific) and PAC1 (GPIIb/IIIa complex-specific) (Fig. 1B). Although antibody OKM5 has been reported to activate platelets,40,41 OKM5 did not result in platelet activation in our whole blood system, as demonstrated by the lack of binding of S12 and PAC1 to OKM5-positive cells in assays performed without thrombin (Fig. 1B).

Downloaded by: NYU. Copyrighted material.

Thrombin Induces Down-Regulation of the Platelet Surface GPIb/IX Complex in the Physiologic Milieu of Whole Blood

Fig. 1. Effect of α-thrombin on the binding of monoclonal antibodies to the platelet surface, as determined by flow cytometry of whole blood. Diluted whole blood in the pres­ ence of 2.5 mM GPRP, a saturating concentration of a fluores­ cein isothiocyanate (FITC)-conjugated GPIV-specific mono­ clonal antibody (OKM5), and a saturating concentration of a biotinylated monoclonal antibody (6D1, FMC25, AK1, S12, or PAC1) was incubated at 22°C with or without purified human α-thrombin (15 minutes), then phycoerythrin-streptavidin (15 minutes), and then an equal volume of 2% formaldehyde (30 minutes). Platelets were identified by their OKM5-positivity (green fluorescence) and their characteristic light scatter. The binding of the biotinylated antibodies was determined from the phycoerythrin (red) fluorescence of the platelets. Panel A: Antibodies 6D1, FMC25, and AK1 are directed against GPIb, GPIX, and the GPIb-IX complex, respectively. For each anti­ body in panel A, the fluorescence intensity of resting platelets was assigned 100 U. Panel B: Antibodies S12 and PAC1 are directed against GMP-140 and the GPIIb-llla complex, respec­ tively. For each antibody in panel B, the fluorescence inten­ sity of maximally activated platelets was assigned 100 U. Data from panels A and B were obtained from the same three experiments (mean ± SEM). (Reproduced with permission from Michelson et al.15)

20

SEMINARS IN THROMBOSIS AND HEMOSTASIS—VOLUME 18, NO. 1, 1992

The blood emerging from a standardized bleeding time wound has been used to assess both soluble and cellular components of the hemostatic system. 42-45 This method has been employed to study platelet activation by indirect means, that is, plasma concentrations of P-thromboglobulin, platelet factor 4, and thromboxane B 2 . 4 3 , 4 4 In a recent study, Abrams et al. 45 directly detected the presence of activated platelets in whole blood emerging from a bleeding time wound. After identifying platelets in whole blood with a GPIb-specific monoclonal antibody, these investigators45 detected activated platelets by increased binding of monoclonal antibodies PAC1 (directed at the fibrinogen receptor on the GPIIb/IIIa complex), 9F9 (specific for fibrinogen bound to the surface of activated platelets), and S12 (GMP-140-specific). As described for assays with thrombin, a modification of the method of Abrams et al. 45 enabled us to study the effect of the standardized bleeding time on the GPIb/IX complex15 (Fig. 2). There was a time-dependent decrease in the platelet surface expression of the GPIb-IX complex, as determined by the binding of monoclonal antibodies AK1 (GPIb-IX complex-specific) and FMC25 (GPIX-specific) (Fig. 2A). Similar results were obtained with GPIb-specific monoclonal antibodies (6D1 and AK3) directed against different epitopes.19,46 As reported by Abrams et al, 45 the bleeding time wound also resulted in a time-dependent increase in the platelet surface expression of GMP-140 (Fig. 2B). Use of a whole blood assay made it very unlikely that the down-regulation of the platelet surface GPIb/IX complex observed during the bleeding time was artifactual.15,33,45

Thrombin-lnduced Down-Regulation of the Platelet Surface GPIb-IX Complex Results in Decreased Binding of von Willebrand Factor George and Torres13 demonstrated that thethrombin-induced decrease in binding of a monoclonal antibody directed at the von Willebrand factor binding site on GPIb was due to a decreased number of binding sites and not to a change in antibody affinity. Furthermore, these investigators13 demonstrated that thrombin stimulation of platelets decreases the binding of von Willebrand factor to GPIb and decreases ristocetin-induced platelet agglutination in vitro reactions that correlate with initial platelet adhesion to the damaged blood vessel wall.

Fig. 2. Time-dependent changes in the platelet surface GPIb-IX complex (panel A) and GMP-140 (panel B) in whole blood emerging from a standardized in vivo bleeding time wound. Whole blood flow cytometry was performed as described in Figure 1, but without the addition of thrombin or GPRP. (For further details see Michelson et al.15) The GPIb-IX complex, GPIX, and GMP-140 were detected by monoclonal antibodies AK1, FMC25, and S12, respectively. The experiment is representative of 15 so performed (Reproduced with permission from Michelson et al.15)

The Thrombin-lnduced Down-Regulation of the Platelet Surface GPIb-IX Complex not Confined to the von Willebrand Factor Binding Site on GPIb The down-regulation of the GPIb-IX complex is not confined to the von Willebrand factor binding site on GPIb, because thrombin induces a parallel decrease in the platelet surface binding of multiple monoclonal antibodies directed against different epitopes on the GPIb-IX complex 12,14,15,30 (Fig. 1 A). Likewise, an in vivo wound results in down-regulation of platelet surface GPIb (data not shown), GPIX (Fig. 2A), and the GPIb-IX complex (Fig. 2A).

Downloaded by: NYU. Copyrighted material.

Down-Regulation of the Platelet Surface GPIb/IX Complex Occurs in Vivo in Response to a Wound

DOWN-REGULATION OF GLYCOPROTEIN COMPLEX—MICHELSON

Despite the marked thrombin-induced changes in the platelet surface expression of GPIb and GPIX, there is maintenance of the approximately 1 : 1 ratio of the binding of a GPIb-specific antibody (6D1) and a GPIXspecific antibody (FMC25) at all thrombin concentra­ tions (Fig. 1A). Furthermore, the parallel binding of AK1 both in vitro (Fig. 1A) and in vivo (Fig. 2A) sug­ gests that the GPIb and GPIX remaining on the plasma membrane of activated platelets are fully complexed, given that AK1 only binds to the intact GPIb-IX complex not to uncomplexed GPIb or GPIX.17

Activation-Induced Down-Regulation of the Platelet Surface GPIb-IX Complex Not Restricted to a Subpopulation of Platelets Because each platelet is analyzed individually, the flow cytometric methods of analyzing platelet surface glycoproteins is able to detect distinct subpopulations of platelets.23,47 In the physiologic setting of whole blood, we therefore addressed the question: When a population of platelets is partially thrombin-activated, is a subpopu­ lation of platelets activated or are all platelets partially activated? As illustrated in Figure 3 for antibodies 6D1 (GPIb-specific) and S12 (GMP-140-specific), thrombin resulted in a concentration-dependent shift of a single peak (to the left for 6D1; to the right for S12). Similar results were obtained (data not shown) with antibodies FMC25 (GPIX-specific) and AK1 (GPIb-IX complexspecific) (shifts to the left) and PAC1 (GPIIb-IIIa com­

Fig. 3. Effect of α-thrombin on platelet surface GPIb and GMP-140, as determined by flow cytometry of whole blood. The assay was performed as described in Figure 1. GPIb (left panel) and GMP-140 (right panel) were detected by mono­ clonal antibodies 6D1 and S12, respectively. For each histo­ gram, the final concentration of thrombin (U/ml) is indicated by the arrows. Each histogram depicts data obtained from 10,000 individual platelets. The experiment is representative of three so performed. (Reproduced with permission from Michelson et al.15)

plex-specific) (shift to the right). Thus, evidence of thrombin activation was not restricted to a distinct subpopulation of platelets, irrespective of whether there was partial or complete activation of platelets. Likewise, the changes in the GPIb-IX complex, the GPIIb-IIIa com­ plex, and GMP-140 induced by the bleeding time wound were not restricted to distinct subpopulations of platelets, as was evident from the shift of a single peak to the left for GPIb-IX-specific monoclonal antibodies15 and to the right for GPIIb-IIIa complex-specific and GMP-140-specific monoclonal antibodies.15,45

On Each Individual Platelet in Whole Blood, Thrombin-induced Surface Exposure of GMP-140 (Degranulation) Is Nearly Complete at the Time of Initiation of Down-Regulation of Surface GPIb George and Torres13 demonstrated that thrombininduced up-regulation of platelet surface GMP-140 pre­ ceded down-regulation of GPIb. However, these authors used a radioligand binding assay of washed platelets that provided an averaged result for all platelets. In order to examine the temporal relationship between the thrombininduced changes in GMP-140 and the GPIb-IX complex on the surface of individual platelets in whole blood, we developed a double-labeling flow cytometric method to simultaneously analyze GMP-140 and GPIb.15 In these double labeling experiments, the binding of two mono­ clonal antibodies (S12 and 6D1) was simultaneously an­ alyzed on each platelet in whole blood by the use of two fluorophores (phycoerythrin and fluorescein isothiocyanate [FITC]) (Fig. 4). On each individual platelet, thrombin-induced degranulation (as monitored by plate­ let surface expression of GMP-140) was nearly complete at the time that down-regulation of surface GPIb was initiated (Fig. 4). Degranulation began within 10 seconds of exposure to thrombin and was nearly complete within 20 seconds (Fig. 4). In contrast, the down-regulation of the platelet surface expression of the GPIb-IX complex did not begin until about 30 seconds after exposure to thrombin and was complete by about 4.5 minutes (Fig. 4). The kinetics of the thrombin-induced decreases in binding of antibodies FMC25 (GPIX-specific) and AK1 (GPIb-IX complex-specific) paralleled the decrease in binding of antibody 6D1 (GPIb-specific) (data not shown).

Thrombin-induced Down-Regulation of Platelet Surface GPIb-IX Complex Not the Result of Cleavage of Platelet Surface GPIb A number of proteases (such as calcium-dependent protease,48 Serratia marcescens protease,49 plasmin,50

Downloaded by: NYU. Copyrighted material.

Fully Complexed GPIb and GPIX Remaining on the Surface of Platelets Activated In Vivo or In Vitro

21

SEMINARS IN THROMBOSIS AND HEMOSTASIS—VOLUME 18, NO. 1, 1992 Some studies13,53 suggest that platelet surface GPIb is still present on the platelet surface after thrombin acti­ vation but that its accessibility to multiple probes is greatly decreased, presumably as a result of either the reported thrombin-induced marked clustering of GPIb molecules on the platelet surface54'55 or a major confor­ mational change in the GPIb-IX complex. The latter is less likely, because such a change would not be expected to result in the same decrease in binding of multiple monoclonal antibodies directed at different epitopes on the.GPIb-IX complex,12,15 and many of these antibodies recognize their epitope on immunoblots, suggesting that they are relatively insensitive to conformational changes in the antigen. Recently, an immunoelectron microscopic study14 reported that the thrombin-induced decrease in the platelet surface GPIb-IX complex was not the result of clustering of GPIb molecules on the platelet surface but was the result of a redistribution of GPIb-IX com­ plexes to the membranes of the open surface canalicular system.

Thrombin-induced Down-Regulation of the Platelet Surface GPIb-IX Complex Mediated via the Platelet Cytoskeleton

Fig. 4. Kinetics of thrombin-induced modulation of platelet surface GPIb and GMP-140, as determined by double labeling of individual platelets in whole blood. Whole blood was incu­ bated at 37°C with α-thrombin 5 U/ml in the presence of 2.5 mM GPRP and the reaction was stopped at various time points by the addition of 1% formaldehyde. The samples were diluted and incubated with a saturating concentration of both FITC-conjugated 6D1 (GPIb-specific) and biotinylated S12 (GMP-140-specific), then incubated with phycoerythrinstreptavidin. Individual platelets were identified by their lightscattering properties, and their fluorescein isothiocyanate and phycoerythrin fluorescence were simultaneously ana­ lyzed by flow cytometry. The density of the dots is directly proportional to the number of platelets. Note that in the up­ permost panel (0 sec) the dots are clustered in the top left quadrant against the ordinate, whereas in the lower three panels (1.5, 4.5, and 10 minutes) the dots are clustered in the bottom right quadrant against the abscissa. The experiment is representative of three so performed. (Reproduced with permission from Michelson et al.15)

and neutrophil elastase51) have been demonstrated to cleave platelet surface GPIb, with resultant release of the proteolytic fragment glycocalicin. However, thrombin does not cleave GPIb.52 Thus, despite the marked throm­ bin-induced decrease in the platelet surface expression of GPIb, there is neither a thrombin-induced decrease in the total platelet content of GPIb 12,13 nor a thrombin-induced release of glycocalicin from the platelet.12

In experiments performed in a washed platelet sys­ tem,15 cytochalasin B specifically inhibited in a concen­ tration-dependent manner the thrombin-induced downregulation of both GPIb and GPIX without inhibiting degranulation (as determined by platelet surface expres­ sion of GMP-140) (Fig. 5). Cytochalasin D had a similar effect.15 George and Torres13 reported similar results with respect to GPIb. These investigators also demon­ strated that cytochalasin prevented the thrombin-induced decrease in von Willebrand factor binding and ristocetininduced platelet agglutination.13 Given that cytochalasins specifically inhibit actin polymerization,56 these data suggest that actin polymerization plays a role in the thrombin-induced down-regulation of the GPIb-IX com­ plex. This is consistent with the known linkage of the GPIb-IX complex to the platelet cytoskeleton via actinbinding protein.21,22

Thrombin-induced Down-Regulation of the GPIb-IX Complex Can Occur via both GPIb-Dependent and GPIb-lndependent Pathways GPIb is a receptor for thrombin.16,29 In order to examine the role of this receptor in the thrombin-induced down-regulation of the GPIb-IX complex, we utilized S. marcescens protease, which selectively cleaves the gly­ cocalicin portion of GPIb.49 Treatment of washed plate­ lets with 2.5 µg/ml S. marcescens protease resulted in

Downloaded by: NYU. Copyrighted material.

22

23

Fig. 5. Effect of cytochalasin B on thrombin-induced down-regulation of GPIb and GPIX on washed platelets. Various concen­ trations of cytochalasin B in 0.4% dimethylsulfoxide were added to washed platelets in modified Tyrode's buffer, pH 7.3 with 5 mM ethylene diaminetetracetic acid prior to activation by 1 U/ml α-thrombin. For antibodies 6D1 (GPIb-specific) and FMC25 (GPIX-specific), the fluorescence intensity of resting platelets in the absence of cytochalasin was assigned 100 U. For antibody S12 (GMP-140-specific), the fluorescence intensity of maximally activated platelets in the absence of cytochalasin was assigned 100 U. Data are mean ± SEM, n = 3. (Reproduced with permission from Michelson et al.15)

virtually complete cleavage of the glycocalicin portion of platelet surface GPIb, as determined by multiple glycocalicin-specific monoclonal antibodies (Fig. 6). In partic­ ular, Serratia protease completely removed the platelet binding site of monoclonal antibody TM60 (Fig. 6). TM60 is directed against the thrombin binding site on the amino-terminal domain of GPIbα. 57-59 In contrast, plate­ let surface GPIX and the GPIIb-IIIa complex were not cleaved by Serratia protease (Fig. 6). Platelet surface GPIb and GPIX also remained complexed after Serratia protease treatment, as was evident from the continued binding of the complex-specific monoclonal antibody AK1 (Fig. 6). Thus, Serratia protease treatment selec­ tively cleaved the glycocalicin portion of platelet surface GPIb without reducing the platelet surface expression of GPIX or the complexing of GPIb and GPIX. Serratia protease did not cause platelet activation, as demon­ strated by the binding of antibodies S12, FMC25, AK1, and 10E5 (Fig. 6). This Serratia protease treatment of platelets did not modify high-dose (10 nM) thrombin-induced up-regulation of GMP-140 or down-regulation of GPIX and the GPIb-IX complex (Fig. 7). In contrast, with low-dose (0.5 nM) thrombin, Serratia protease treatment abol­ ished the up-regulation of platelet surface GMP-140, while diminishing the down-regulation of platelet surface GPIX by 60.9 ± 5.6% (mean ± SEM, n = 3) and the GPIb-IX complex by 60.2 ± 0.5% (Fig. 7).

In summary, low-dose but not high-dose thrombin requires the presence of the glycocalicin portion of plate­ let surface GPIb to stimulate alpha granule secretion (as determined by GMP-140) and maximal down-regulation of the platelet surface GPIb-IX complex. These studies suggest: (1) The high affinity receptor for thrombin is on the glycocalicin portion of GPIb; and (2) platelets have an additional moderate affinity receptor for thrombin, not on the glycocalicin portion of GPIb, that is sufficient to induce a granule secretion and maximal down-regulation of the GPIb-IX complex. Thus, thrombin-induced downregulation of the GPIb-IX complex can occur via both GPIb-dependent and GPIb-independent pathways.30

Thrombin Requires an Active Catalytic Site to Induce Down-Regulation of the GPIb-IX Complex. Thrombin requires an active catalytic site to induce down-regulation of the GPIb-IX complex, because the down-regulation is not observed with thrombin inacti­ vated by diisopropyl fluorophosphate (DFP)11,13 or D-phenylalanine-L-prolyl-L-arginine chloromethyl ke­ tone (PPACK).14

Downloaded by: NYU. Copyrighted material.

DOWN-REGULATION OF GLYCOPROTEIN COMPLEX—MICHELSON

SEMINARS IN THROMBOSIS AND HEMOSTASIS—VOLUME 18, NO. 1, 1992

Fig. 6. Effect of Serratia protease on platelet surface glycoproteins. Washed platelets were incubated (30 minutes, 22°C) with or without Serratia protease, fixed, washed, incubated with a saturating concentration of a fluorescent-labeled monoclonal antibody, and analyzed by flow cytometry. For antibodies 6D1, TM60, AK3, FMC25, AK1, and 10E5, the fluorescence intensity of non-Serratia protease-treated platelets was assigned 100 U. For antibody S12, the fluorescence intensity of maximally activated (thrombin 10 nM) non-Serratia protease-treated platelets was assigned 100 U (see Fig. 7). Glycocalicin release from the platelet surface by Serratia protease was measured by enzyme-linked immunosorbent assay using 6D1. Data are mean ± SEM, n = 3. (Reproduced with permission from Yamamoto et al.30)

Other Agonists Inducing Down-Regulation of Platelet Surface Expression of the GPIb-IX Complex Adenosine diphosphate (ADP) also induces downregulation of the platelet surface expression of the GPIb-IX complex.15 In a whole blood system, the maximal ADP-induced decrease in the binding of monoclonal antibodies directed against the GPIb-IX complex was approximately 50%. 15 Hourdillé et al. 14 detected a more modest ADP-induced down-regulation of GPIb, but these investigators used a washed platelet system, which has the potential problem of platelet refractoriness.60 Collagen and epinephrine have minor effects on the platelet surface binding of a GPIb-specific monoclonal antibody.15 A maximal decrease of 32.0 ± 2.5% (mean ± SEM, n = 3) occurred with 100 µg/ml collagen.15 A maximal decrease of 14.0 ± 5.7% occurred with 10 µM epinephrine.15 The calcium ionophore A23187 does not down-regulate GPIb, 13,14 presumably because A23187 hydrolyzes actin-binding protein. Plasmin at concentrations 1 CU/ml or higher has been reported to activate platelets.61 Plasmin 1 CU/ml has been demonstrated to result in an increase in platelet

surface GMP-140 and a decrease in platelet surface GPIb. 62 In contrast, plasmin at concentrations < 1 CU/ml has been reported to inhibit platelet activation in response to thrombin and other agonists63 and to cleave platelet surface GPIb. 50,64-66 However, it was recently reported that plasmin 0.2 CU/ml resulted in a decrease in platelet surface GPIb that was not associated with loss of glycocalicin from the platelets but was associated with degranulation and a redistribution of GPIb to the surface-connected canalicular system.67,68 Under apparently similar conditions, we 66 observed a plasmin-induced loss of platelet surface GPIb that was associated with loss of glycocalicin from the platelets but not with degranulation.

Platelet Degranulation and the Activation-Dependent Down-Regulation of Platelet Surface GPIb-IX Complex as Independent Processes The fact that thrombin-induced degranulation was nearly complete at the time that down-regulation of the platelet surface GPIb-IX complex was initiated (Fig. 4) raises the question as to whether platelet surface exposure

Downloaded by: NYU. Copyrighted material.

24

25

Fig. 7. Effect of thrombin on the platelet surface GPIb-IX complex and GMP-140 on Serratia protease-treated platelets, as determined by flow cytometry. Washed platelets were incubated for 30 minutes at 22°C with ("SP") or without ("C") Serratia protease 2.5 µg/ml, washed, and incubated for 10 minutes at 37°C with 0,0.5, or 10 nM purified human α-thrombin. For antibodies FMC25, AK1, 6D1, and AK3, the fluorescence intensity of platelets incubated with neither Serratia protease nor thrombin was assigned 100 U. For antibody S12, the fluorescence intensity of platelets incubated without Serratia protease but maximally activated with thrombin (10 nM) was assigned 100 U. Data are mean ± SEM, n = 3. (Reproduced with permission from Yamamoto et al.30)

of GMP-140, or release of another alpha granule compo­ nent or components, is a prerequisite for activation-de­ pendent GPIb-IX down-regulation. However, three lines of evidence demonstrate that down-regulation of platelet surface GPIb-IX complex and degranulation can be un­ coupled: (1) ADP induces down-regulation of the GPIb-IX complex without significant exposure of GMP140 on the surface of the same platelets;15 (2) cytochalasins inhibit the down-regulation of platelet surface GPIb-IX but not the up-regulation of platelet surface GMP-14013 (Fig. 5); and (3) in bleeding time experi­ ments in vivo, up-regulation of platelet surface GMP-140 was not complete at the time that down-regulation of platelet surface GPIb-IX was initiated (Fig. 2).

PLASMIN-INDUCED AND STORAGEINDUCED REDISTRIBUTION OF GLYCOPROTEIN lb In contrast to the effect of thrombin just described, we have provided evidence that both in vitro platelet storage23 and plasmin66 can result in a redistribution of

GPIb molecules in the opposite direction: intraplatelet pool, to platelet surface pool, to plasma glycocalicin pool. Thus, GPIb molecules can traffic between different pools.

CONCLUSIONS Thrombin activation of platelets results in downregulation of the GPIb-IX complex. This down-regula­ tion results in decreased accessibility of the von Willebrand factor binding site on GPIb. In contrast, thrombin induces up-regulation of the GPIIb-IIIa complex, result­ ing in exposure of receptors for fibrinogen,4 von Willebrand factor,5 fibronectin,6 and vitronectin.7 Thus, thrombin transforms the platelet from a proadhesive state (mediated by the GPIb-IX complex) to a proaggregatory state (mediated by the GPIIb-IIIa complex). Down-regulation of the GPIb-IX complex may be a useful marker of platelet activation in vivo. We are ac­ tively investigating this possibility. Acknowledgments. Supported by Grant No. HL38138 from the National Institutes of Health. For the experiments

Downloaded by: NYU. Copyrighted material.

DOWN-REGULATION OF GLYCOPROTEIN COMPLEX—MICHELSON

SEMINARS IN THROMBOSIS AND HEMOSTASIS—VOLUME 18, NO. 1, 1992

performed in my laboratory, I thank Dr. John W. Fenton II (New York Department of Health, Albany) for generously pro­ viding purified human α-thrombin; Drs. Michael C. Berndt (University of Sydney, Australia), Barry S. Coller (SUNY, Stony Brook), Rodger P. McEver (University of Oklahoma), Sanford J. Shattil (University of Pennsylvania, Philadelphia), and Naomasa Yamamoto (Tokyo Metropolitan Institute of Medical Science) for generously providing antibodies; Drs. G.A. Jamieson and Peter H. Levine for their support; and Dr. Anita S. Kestin, Patricia A. Ellis, and Marc R. Barnard for their essential contributions.

REFERENCES 1. Berndt MC, C Gregory, G Dowden, PA Castaldi: Thrombin inter­ actions with platelet membrane proteins. Ann NY Acad 374-386, 1986. 2. Hansen SR, LA Harker: Interruption of acute platelet-dependent thrombosis by the synthetic antithrombin PPACK. Proc Natl Acad Sci USA 85:3184-3188, 1988. 3. Eidt JF, P Allison, S Nobel J, Ashton, P Golino, J McNatt, LM Buja, JT Willerson: Thrombin is an important mediator of platelet aggression in stenosed canine coronary arteries with endothelial injury. J Clin Invest 84:18-27, 1989. 4. Shattil SJ, JA Hoxie, M Cunningham, LF Brass: Changes in the platelet membrane glycoprotein IIb-IIIa complex during platelet activation. J Biol Chem 260:11107-11114, 1985. 5. Ruggeri ZM, L De Marco, L Gatti, R Bader, RR Montgomery: Platelets have more than one binding site for von Willebrand fac­ tor. J Clin Invest 72:1-12, 1983. 6. Plow EF, MH Ginsberg: Specific and saturable binding of plasma fibronectin to thrombin-stimulated human platelets. J Biol Chem 256:9477-9482, 1981. 7. Asch E, E Podack: Vitronectin binds to activated human platelets and plays a role in platelet aggregation. J Clin Invest 85:13721378, 1990. 8. Stenberg PE, RP McEver, MA Shuman, YV Jacques, DF Bainton: A platelet alpha-granule membrane protein (GMP-140) is ex­ pressed on the plasma membrane after activation. J Cell Biol 101:880-886, 1985. 9. Febbraio M, RL Silverstein: Identification and characterization of LAMP-1 as an activation-dependent platelet surface glycoprotein. J Biol Chem 265:18531-18537, 1990. 10. Nieuwenhuis HK, JJ van Oosterhout, E Rozemuller, F van Iwaarden, JJ Sixma: Studies with a monoclonal antibody against acti­ vated platelets: Evidence that a secreted 53,000-molecular weight lysosome-like granule protein is exposed on the surface of acti­ vated platelets in the circulation. Blood 70:838-845, 1987. 11. George JN, EB Pickett, S Saucerman, RP McEver, TJ Kunicki, N Kieffer, PJ Newman: Platelet surface glycoproteins. Studies on resting and activated platelets and platelet membrane microparticles in normal subjects, and observations in patients during adult respiratory distress syndrome and cardiac surgery. J Clin Invest 78:340-348,1986. 12. Michelson AD, MR Barnard: Thrombin-induced changes in plate­ let membrane glycoproteins Ib, IX, and IIb-IIIa complex. Blood 70:1673-1678, 1987. 13. George JN, MM Torres: Thrombin decreases von Willebrand fac­ tor binding to platelet glycoprotein Ib. Blood71:1253-1259, 1988. 14. Hourdillé P, Heilmann, R Combrie, J Winckler, KJ Clemetson, AT Nurden: Thrombin induces a rapid redistribution of glycoprotein Ib-IX complexes within the membrane systems of activated human platelets. Blood 76:1503-1513, 1990.

15. Michelson AD, PA Ellis, MR Barnard, GB Matic, AF Viles, AS Kestin: Down-regulation of the platelet surface glycoprotein Ib-IX complex in whole blood stimulated by thrombin, ADP or an in vivo wound. Blood 77:770-779, 1991. 16. Ruggeri ZM: The platelet glycoprotein Ib-IX complex. Prog Hemost Thromb 10:35-68, 1991. 17. Du X, L Beutler, C Ruan, PA Castaldi, MC Berndt: Glycoprotein Ib and glycoprotein IX are fully complexed in the intact platelet membrane. Blood 69:1524-1527, 1987. 18. Roth GJ: Developing relationships: arterial platelet adhesion, glycoprotein Ib, and leucine-rich glycoproteins. Blood 77:5-19, 1991. 19. Coller BS, EI Peerschke, LE Scudder, CA Sullivan: Studies with a murine monoclonal antibody that abolishes ristocetin-induced binding of von Willebrand factor to platelets: Additional evidence in support of GPIb as a platelet receptor for von Willebrand factor. Blood 61:99-110, 1983. 20. Phillips DR, IF Charo, LV Parise, LA Fitzgerald: The platelet membrane glycoprotein IIb-IIIa complex. Blood 71:831-843, 1988. 21. Fox JEB: Identification of actin-binding protein as the protein linking the membrane skeleton to glycoproteins on platelet plasma membranes. J Biol Chem 260:11970-11977, 1985. 22. Fox JEB: Linkage of a membrane skeleton to integral membrane glycoproteins in human platelets. Identification of one of the glycoproteins as glycoprotein Ib. J Clin Invest 76:1673-1683, 1985. 23. Michelson AD, B Adelman, MR Barnard, E Carroll, RI Handin: Platelet storage results in a redistribution of glycoprotein Ib molecules. Evidence for a large intraplatelet pool of glycoprotein Ib. J Clin Invest 81:1734-1740, 1988. 24. Wencel-Drake JD, EF Plow, TJ Kunicki, VL Woods, DM Keller, MH Ginsberg: Localization of internal pools of membrane glycoproteins involved in platelet adhesive responses. Am J Pathol 124:324-334, 1986. 25. Asch AS, LLK Leung, MJ Polley, RL Nachman: Platelet membrane topography: Colocalization of thrombospondin and fibrinogen with the glycoprotein IIb-IIIa complex. Blood 66:926-934, 1985. 26. Coller BS, E Kalomiris, M Steinberg, LE Scudder: Evidence that glycocalicin circulates in normal plasma. J Clin Invest 73:794799, 1984. 27. Okumura T, C Lombart, GA Jamieson: Platelet glycocalicin. II. Purification and characterization. J Biol Chem 251:5950-5955, 1976. 28. Michelson AD, J Loscalzo, B Melnick, BS Coller, RI Handin: Partial characterization of a binding site for von Willebrand factor on glycocalicin. Blood 67:19-26, 1986. 29. Jamieson GA: The activation of platelets by thrombin: A model for activation by high and moderate affinity receptors In: Jamieson GA: Platelet Membrane Receptors: Molecular Biology, Immunology, Biochemistry, and Pathology. Alan R. Liss, New York, 1988, pp 137-158. 30. Yamamoto N, NJ Greco, MR Barnard, K Tanoue, H Yamazaki, GA Jamieson, AD Michelson: Glycoprotein Ib (GPIb)-dependent and GPIb-independent pathways of thrombin-induced platelet activation. Blood 77:1740-1748, 1991. 31. Vu T-KH, DT Hung, VI Wheaton, SR Coughlin: Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 64:1057-1068, 1991. 32. Fox JE, MC Berndt: Cyclic AMP-dependent phosphorlyation of glycoprotein Ib inhibits collagen-induced polymerization of actin in platelets. J Biol Chem 264:9520-9526, 1989. 33. Shattil SJ, M Cunningham, JA Hoxie: Detection of activated platelets in whole blood using activation-dependent monoclonal antibodies and flow cytometry. Blood 70:307-315, 1987.

Downloaded by: NYU. Copyrighted material.

26

34. Laudano AP, RF Doolittle: Synthetic peptide derivatives that bind fibrinogen and prevent the polymerization of fibrin monomers. Proc Natl Acad Sci USA 75:3085-3089, 1978. 35. Laudano AP, RF Doolittle: Studies on synthetic peptides that bind to fibrinogen and prevent fibrin polymerization. Structural require­ ments, number of binding sites, and species differences. Biochem­ istry 19:1013-1019, 1980. 36. Laudano AP, RF Doolittle: Influence of calcium ion on the binding of fibrin amino terminal peptides to fibrinogen. Science 212:457459,1981. 37. Asch AS, J Barnwell, RL Silverstein, RL Nachman: Isolation of the thrombospondin membrane receptor. J Clin Invest 79:1054— 1061, 1987. 38. Takamatsu J, MK Horne, HK Gralnick: Identification of the thrombin receptor on human platelets by chemical crosslinking. J Clin Invest 77:362-368, 1986. 39. Harmon JT, GA Jamieson: The glycocalicin portion of platelet glycoprotein Ib expresses both high and moderate affinity receptor sites for thrombin. J Biol Chem 261:13224-13229, 1986. 40. Ockenhouse CF, C Magowan, JD Chulay: Activation of mono­ cytes and platelets by monoclonal antibodies or malaria-infected erythrocytes binding to the CD36 surface receptor in vitro. J Clin Invest 84:468-475, 1989. 41. Aiken ML, MH Ginsberg, V Byers-Ward, EF Plow: Effects of OKM5, a monoclonal antibody to glycoprotein IV, on platelet aggregation and thrombospondin surface expression. Blood 76:2501-2509,1990. 42. Novotny WF, TJ Girard, JP Miletich, GJ Broze, Jr.: Platelets secrete a coagulation inhibitor functionally and antigenically simi­ lar to the lipoprotein associated coagulation inhibitor. Blood 72:2020-2025, 1988. 43. Weiss HJ, B Lages: Evidence for tissue factor-dependent activa­ tion of the classic extrinsic coagulation mechanism in blood ob­ tained from bleeding time wounds. Blood 71:629-635, 1988. 44. Kyrle PA, F Stockenhuber, B Brenner, H Gossinger, C Korninger, I Pabinger, G SunderPlassman, P Balcke, K Lechner: Evidence for an increased generation of prostacyclin in the microvasculature and an impairment of the platelet α-granule release in chronic renal failure. Thromb Haemost 60:205-208, 1988. 45. Abrams CS, N Ellison, AZ Budzynski, SJ Shattil: Direct detection of activated platelets and platelet-derived microparticles in hu­ mans. Blood 75:128-138, 1990. 46. Berndt MC, X Du, WJ Booth: Ristocetin-dependent reconstitution of binding of von Willebrand factor to purified human platelet membrane glycoprotein Ib-IX complex. Biochemistry 27:633640, 1988. 47. Michelson AD: Flow cytometric analysis of platelet surface glyco­ proteins: Phenotypically distinct subpopulations of platelets in children with chronic myeloid leukemia. J Lab Clin Med 110:346354,1987. 48. McGowan EB, K-T Yeo, TC Detwiler: The action of calciumdependent protease on platelet surface glycoproteins. Arch BiochemBiophys 227:287-301, 1983. 49. Cooper HA, WP Bennett, GC White, RH Wagner: Hydrolysis of human platelet membrane glycoproteins with a Serratia marcescens metalloprotease: Effect on response to thrombin and von Willebrand factor. Proc Natl Acad Sci USA 79:1433-1437, 1982. 50. Adelman B, AD Michelson, J Greenberg, RI Handin: Proteolysis of platelet glycoprotein Ib by plasmin is facilitated by plasmin lysine-binding regions. Blood 68:1280-1284, 1986.

27

51. Brower MS, RI Levin, K Garry: Human neutrophil elastase modu­ lates platelet function by limited proteolysis of membrane glyco­ proteins. J Clin Invest 75:657-666, 1985. 52. Berndt MC, DR Phillips: Purification and preliminary physicochemical characterization of human platelet membrane glycopro­ tein V. J Biol Chem 256:59-65, 1981. 53. George JN, RM Lyons, RK Morgan: Membrane changes associ­ ated with platelet activation. Exposure of actin on the platelet surface after thrombin-induced secretion. J Clin Invest 66:1-9, 1980. 54. Polley MJ, LL Leung, FY Clark, RL Nachman: Thrombin-induced platelet membrane glycoprotein lib and Ilia complex formation. An electron microscope study. J Exp Med 154:1058-1068, 1981. 55. Suzuki H, N Yamamoto, K Tanoue, H Yamazaki: Glycoprotein lb distribution on the surface of platelets in resting and activation states: An electron microscope study. Histochem J 19:125-136, 1987. 56. Fox JEB, DR Phillips: Inhibition of actin polymerization in blood platelets by cytochalasins. Nature 292:650-652, 1981. 57. Yamamoto N, H Kitagawa, K Tanoue, H Yamazaki: Monoclonal antibody to glycoprotein Ib inhibits both thrombin- and ristocetininduced platelet aggregations. Thromb Res 39:751-759, 1985. 58. Yamamoto K, N Yamamoto, H Kitagawa, K Tanoue, G Kosaki, H Yamazaki: Localization of a thrombin-binding site on human plate­ let membrane glycoprotein Ib determined by a monoclonal anti­ body. Thromb Haemost 55:162-167, 1986. 59. Katagiri Y, Y Hayashi, K Yamamoto, K Tanoue, G Kosaki, H Yamazaki: Localization of von Willebrand factor and thrombininteractive domains on human platelet glycoprotein Ib. Thromb Haemost 63:122-126, 1990. 60. Kinlough-Rathbone RL, MA Packham, JF Mustard: Platelet ag­ gregation In: Harker LA, TS Zimmerman: Measurements of Platelet Function. Churchill Livingstone, New York, 1983, pp 64—91. 61. Schafer AI, AK Mass, JA Ware, PC Johnson, SE Rittenhouse, EW Salzman: Platelet protein phosphorylation, elevation of cytosolic calcium, and inositol phospholipid breakdown in platelet activa­ tion induced by plasmin. J Clin Invest 78:73-79, 1986. 62. Bachmann F, P Parise: Fibrinolysis. In: Friedel N, R Hetzer, D Royston: Blood Use in Cardiac Surgery. Steinkopf Verlag, Darm­ stadt, 1991, pp 3-9. 63. Schafer AI, B Adelman: Plasmin inhibition of platelet function and of arachidonic acid metabolism. J Clin Invest 75:456-461, 1985. 64. Adelman B, AD Michelson, J Loscalzo, J Greenberg, RI Handin: Plasmin effect on platelet glycoprotein Ib-von Willebrand factor interactions. Blood 65:32-40, 1985. 65. Stricker RB, D Wong, DT Shiu, PT Reyes, MA Shuman: Activa­ tion of plasminogen by tissue plasminogen activator on normal and thrombasthenic platelets: Effects on surface proteins and platelet aggregation. Blood 68:275-280, 1986. 66. Michelson AD, MR Barnard: Plasmin-induced redistribution of platelet glycoprotein Ib. Blood 76:2005-2010, 1990. 67. Cramer EM, H Lu, JP Caen, C Soria, MC Berndt, D Tenza: Differential redistribution of platelet glycoproteins Ib and IIb-IIIa after plasmin stimulation. Blood 77:694-699, 1991. 68. Lu H, C Soria, EM Cramer, J Soria, J Maclouf, JY Perrot, H Li, PL Commin, F Schumann, O Regnier, RH Lijnen, JP Caen: Tem­ perature dependence of plasmin-induced activation or inhibition of human platelets. Blood 77:996-1005, 1991.

Downloaded by: NYU. Copyrighted material.

DOWN-REGULATION OF GLYCOPROTEIN COMPLEX—MICHELSON