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

Events Occurring After Thrombin-lnduced Fibrinogen Binding to Platelets

The association between fibrinogen and the surface of activated platelets is a prerequisite for platelet aggre­ gation in response to a variety of substances, including adenosine diphosphate (ADP), epinephrine, collagen, and thrombin (reviewed by Peerschke1). Two sources of fibrinogen are thought to be important: extracellular fi­ brinogen and fibrinogen contained in platelet alpha gran­ ules.2 The binding of fibrinogen to thrombin-stimulated platelets was initially characterized in two laborato­ ries. 3,4 To restrict the activity of thrombin to platelets as opposed to fibrinogen, platelets were stimulated with thrombin for 5 to 10 minutes and then excess hirudin was added to neutralize thrombin before the addition of 125 Ifibrinogen. Maximal fibrinogen binding was divalent cation dependent, required approximately 200 µg/ml of added fibrinogen, and occurred within 5 to 15 minutes of platelet stimulation. Approximately 40,000 molecules of fibrinogen bound per platelet with a dissociation constant of 2 x 1 0 - 7 M . Fibrinogen receptors were localized to the platelet membrane glycoprotein IIb-IIIa (GPIIb-IIIa) complex (reviewed by Phillips et al5). This complex is a member of a family of adhesive protein receptors, also known as integrins6 or cytoadhesins.7 The majority of receptors on resting platelets fail to bind fibrinogen. On stimulation, however, the GPIIb-IIIa complex is rendered competent through activation-dependent conformational changes in the receptor itself or its microenvironment.8-10 Depend­ ing on the agonist and its dose, platelet activation may be enhanced by the release of dense granule ADP and syn­

From the Department of Pathology, SUNY at Stony Brook, Stony Brook, New York. Reprint requests: Dr. Peerschke, University Hospital, L-3, SUNY at Stony Brook, Stony Brook, NY 11794-7300. 34

thesis of prostaglandin endoperoxides and thromboxane A2 (reviewed by Peerschke1). Although there is little doubt that released ADP, prostaglandin endoperoxides, and thromboxane A2 potentiate platelet responses, the question of whether agonists such as thrombin, epineph­ rine, and arachidonic acid expose fibrinogen receptors directly is still controversial. Fibrinogen contains several platelet-binding do­ mains. Two RGD sequences are located on each of fi­ brinogen's two Aα chains: amino acids 95-97 and 572574.11 Another platelet recognition sequence is found at the carboxy-terminus of fibrinogen gamma chains, en­ compassing residues gamma 400-411. 12 Unlike the alpha chain RGD sequences, which are shared by other adhe­ sive glycoproteins, the gamma chain platelet-binding do­ main appears to be unique to fibrinogen. RGD and gamma 400-411 dodecapeptide binding sites have been identified on the GPIIb-IIIa complex. Photoaffinity labeling studies indicate that RGD-containing peptides are incorporated into both GPIIb and GPIIIa, whereas gamma chain peptides are incorporated primar­ ily into GPIIb.13 Chemical cross-linking experiments have localized an RGD binding site to amino acids 109171 in GPIIIa14 and a gamma chain binding site to amino acids 294-314 in GPIIb.15 The binding of RGD and gamma chain dodecapeptides to the intact GPIIb-IIIa com­ plex, however, appears to be mutually exclusive, 16-18 suggesting that these sites may be spatially or conformationally related. Although considerable attention has focused on events leading to and supporting platelet interaction with adhesive glycoproteins, little is known about the fate and function of these ligands once bound to the platelet sur­ face. I have been interested in the progressive stabiliza­ tion of platelet fibrinogen interactions, first described by Marguerie et al19 in 1980, and its contribution to platelet physiology and pathology. The following discussion

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

Downloaded by: University of British Columbia. Copyrighted material.

ELLINOR I.B. PEERSCHKE, Ph.D.

35

Fig. 1. Temperature dependence of irreversible fibrinogen binding.20 The data represent a typical experiment.

summarizes the present understanding of post-fibrinogen binding events involved in stabilizing fibrinogen interactions with thrombin-stimulated platelets and their functional implications. It will focus on the biochemical characterization of "irreversible" fibrinogen binding, qualitative changes in fibrinogen binding involving alterations in fibrinogen expression and distribution on the platelet surface, the role of the platelet cytoskeleton and the release reaction, and the role of the GPIIb-IIIa complex and known fibrinogen structural domains.

STABILIZATION OF PLATELET FIBRINOGEN INTERACTIONS: BIOCHEMICAL CHARACTERIZATION General Properties The interaction between fibrinogen and platelets has been described as a multiphasic process culminating in the stabilization of fibrinogen binding to the surface of activated platelets.19 This stabilization is characterized by the failure of bound fibrinogen to be dissociated by excess unlabeled fibrinogen or 5 to 10 mM ethylene diaminetetraacetic acid (EDTA).19,20 Fibrinogen that remains bound to platelets under these conditions is defined

as being "irreversibly" bound19,20 or "EDTA-resistant."21 This phenomenon was initially described using washed platelets stimulated with ADP.19 Similar observations were made later in platelet-rich plasma22 and using platelets stimulated with thrombin or epinephrine.20 The development of irreversible platelet-fibrinogen interactions is time-, 19,20 temperature- (Fig. 1), and agonist-dependent20 (Fig. 2). It is unaffected by the divalent cation composition of the platelet-suspending media, provided sufficient calcium or magnesium is present to support fibrinogen binding (Table 1). Maximal stabilization of platelet-fibrinogen interactions requires the incubation of activated but unaggregated platelets with fibrinogen for 40 to 60 minutes at 22°C.19,20 Only 10 to 20% of ADP- or thrombin-induced fibrinogen binding becomes resistant to dissociation by either EDTA or unlabeled fibrinogen during the first 5 to 10 minutes after platelet exposure to fibrinogen. In contrast, approximately 50% of platelet-bound fibrinogen becomes resistant to dissociation within 60 minutes. The formation of irreversible interactions between platelets and fibrinogen is potentiated at 37°C and diminished at 4°C.20 Interestingly, exposure of fibrinogen receptors by chymotrypsin23 leads to the formation of significantly less irreversible fibrinogen binding compared with platelet stimulation with either ADP or thrombin.20

Downloaded by: University of British Columbia. Copyrighted material.

POST-FIBRINOGEN BINDING EVENTS—PEERSCHKE

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

Fig. 2. Agonist effect on the development of irreversible platelet fibrinogen interactions.20 The data represent a typical experiment.

Role of Platelet Cytoskeleton A role for the platelet cytoskeleton in stabilizing platelet fibrinogen interaction was suggested by the observation that stabilization of fibrinogen binding is attenuated by preincubating platelets with cytochalasins.20 More recently, a time-dependent interaction between fibrinogen and the Triton X-100 insoluble cytoskeleton of activated platelets was also demonstrated and paralleled

development of EDTA-resistant, irreversible fibrinogen binding.21 Maximum incorporation of fibrinogen bound to thrombin-stimulated platelets into the Triton X-100 insoluble cytoskeleton occurred 45 to 60 minutes after platelet activation and fibrinogen binding at 22°C, and comprised 80 to 100% of irreversibly bound fibrinogen.21 Because fibrin can precipitate nonspecifically with the platelet cytoskeleton due to its insolubility in

TABLE 1. Effect of Divalent Cations on Stabilizing Platelet-Fibrinogen Interactions and Bound Fibrinogen Accessibility to Antifibrinogen Antibody* Divalent Cation

Irreversible Fibrinogen Binding (%)

Antifibrinogen Antibody Binding (%)

Magnesium

64 + 10

45 + 11

Calcium

65 ± 12 6 4 + 12

4 0 + 17

Calcium plus magnesium

43+11

*Thrombin-stimulated platelets were suspended in buffer containing 1 mM magnesium chloride, 1 mM calcium chloride, or both, and incubated with either 125I-labeledor unlabeled fibrinogen at 22°C. Irreversible fibrinogen binding and antifibrinogen antibody binding (mean ± SD, n = 5) were quantified or as described.29,30 Briefly, irreversible fibrinogen binding was assessed after adding 10 mM ethylene diamine tetraacetic acid to an aliquot of stimulated platelets that were incubated with labeled fibrinogen for 60 minutes, and comparing fibrinogen remaining bound 30 minutes later with total fibrinogen binding. Antifibrinogen antibody binding was measured using 125I-labeled antibody Fab fragments added to stimulated platelets incubated with unlabeled fibrinogen for 5 and 60 minutes. Antibody binding to platelets exposed to fibrinogen for 60 minutes is expressed as a percent relative to antibody binding to platelets exposed to fibrinogen for 5 minutes.

Downloaded by: University of British Columbia. Copyrighted material.

36

POST-FIBRINOGEN BINDING EVENTS—PEERSCHKE

Contribution of the Release Reaction Compared with platelet stimulation with ADP, stimulation with thrombin leads to enhanced stabilization of platelet-fibrinogen interactions20,21 and enhanced fibrinogen coprecipitation with the activated platelet cytoskeleton.21 Platelet stimulation with thrombin is also accompanied by significant alpha granule release.2 Recent studies suggest that thrombospondin (TSP), an alpha granule constituent, can interact specifically with fibrinogen at sites that are different from those involved in fibrinogen binding to the platelet membrane GPIIb-IIIa complex (reviewed by Asch and Nachman27). Fab fragments prepared from anti-TSP antibodies have been shown to inhibit the second wave of aggregation elicited by ADP, and partially inhibit thrombin and collageninduced aggregation.28 Moreover, the rate of fibrinogen dissociation from the platelet surface is enhanced in the presence of anti-TSP antibodies,28 supporting the hypothesis that TSP interacts with fibrinogen on the platelet surface and helps to stabilize its binding. The interaction between platelet-associated fibrinogen and released TSP, however, is not an absolute requirement for the development of irreversible plateletfibrinogen interactions. Significant irreversible fibrin-

ogen binding is observed following stimulation of aspirin-treated platelets with ADP at 22°C, when little to no alpha granule release is detected.29 Moreover, the extent of irreversible fibrinogen binding to platelets from a patient with the gray platelet syndrome was similar to irreversible fibrinogen binding noted with platelets from a normal volunteer.29

Accessibility of Bound Fibrinogen to Antibody Recent studies have illustrated time-dependent decreases in platelet-bound fibrinogen accessibility to antibody, its F(ab')2, or Fab fragments30 (Table 1). Although no changes in platelet-bound fibrinogen were detected, the amount of antifibrinogen antibody binding to platelets 60 minutes after stimulation with thrombin was less than 50% of that bound to platelets 5 minutes after activation. This phenomenon parallels the development of irreversible platelet fibrinogen interactions30 and demonstrated similar temperature requirements (Fig. 3). Internalization of bound fibrinogen is probably not responsible for the decreased binding of antifibrinogen antibody, since the fibrinogen remains accessible to enzymatic digestion, as described in the next section. Trivial explanations for the observed, decreased fibrinogen accessibility to antibody, such as platelet aggregation and the release of alpha granule fibrinogen stores, were excluded. Since platelet samples were not stirred or shaken mechanically to induce platelet-platelet contact, platelet aggregates were not only macroscopically absent during the course of the experiment, but light microscopic observations also failed to reveal the presence of more than occasional aggregates of three to five platelets with no difference between samples examined 5 or 60 minutes after platelet stimulation. Competition for antibody by released alpha granule fibrinogen was excluded because similar observations were made with aspirintreated platelets stimulated with ADP at 22°C in which there was minimal detectable alpha granule release.29,30 Moreover, similar decreases in accessibility of bound fibrinogen degradation products (early fragment X) and fibrinogen variants (fibrinogen gamma 57.5 or 7'), not present in platelet alpha granules,31,32 were noted using specific monoclonal antibodies (Table 2). New antigenic determinants, referred to as receptorinduced binding sites (RIBS),33 have been detected on the fibrinogen molecule following binding to stimulated platelets.31 Monoclonal antibodies have localized one RIBS to the fibrinogen gamma chain D domain. Conformational changes may contribute to the progressive alteration in bound fibrinogen expression at the platelet surface, but appear unlikely to account entirely for the

Downloaded by: University of British Columbia. Copyrighted material.

nonionic detergents,24 fibrin formation of exogenously added 125I-fibrinogen was prevented by stimulating platelets with thrombin and neutralizing thrombin with hirudin before fibrinogen was added. In addition, studies were performed in the presence of the gly-pro-arg-pro peptide (GPRP), which prevents fibrin assembly,25 and similar results were obtained. Moreover, the recovery of irreversibly bound fibrinogen with platelet cytoskeletons was not unique to thrombin-stimulated platelets. Approximately 50% of fibrinogen that became associated with ADP-treated platelets in an EDTA-resistant manner also coprecipitated with the cytoskeleton.21 Cytoskeleton analysis by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography revealed that the associated 125I-fibrinogen was indistinguishable from native fibrinogen.21 Cytoskeletons of activated but unaggregated platelets were composed predominantly of actin, myosin heavy and light chains, alpha actinin, and actin-binding protein. Consistent with reports demonstrating maximum cytoskeletal formation 5 to 15 minutes after platelet stimulation,26 no changes in individual cytoskeletal proteins were noted during the time required for maximal development of irreversible fibrinogen binding.21 Comparison of cytoskeletons derived from thrombin or ADP-treated platelets, however, showed increases in all cytoskeletal components following platelet stimulation with thrombin.21

37

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

Fig. 3. Results from a typical experiment illustrating the temperature requirements for modulation of platelet-associated fibrinogen accessibility to antifibrinogen antibody Fab fragments. Antifibrinogen Fab binding was quantified as described. 30 Briefly, 125l-labeled Fab fragments were added to thrombin-stimulated platelets that had previously been incubated with fibrinogen for 5 and 60 minutes (22°C). The data express decreases in antifibrinogen Fab binding to platelets incubated with fibrinogen for 60 minutes compared to Fab binding to platelets incubated with fibrinogen for 5 mintites.

dramatic, time-dependent reduction in bound fibrinogen recognition by polyclonal antibodies.30

TABLE 2. Accessibility of Bound Fibrinogen to Antibody: Comparison of Fibrinogen Variants and Degradation Products Antibody Binding Fibrinogen (%)* Antibody

Gamma50

Gamma 57.5†

Fragment X

Polyclonal antifibrinogen

46 + 7

44 + 12

3 8 + 12

Monoclonal L2B

NR§

41 + 10

ND

Monoclonal GC4‡

ND§

ND

4 0 + 13

* Antifibrinogen antibody binding was evaluated as described.30 Briefly 125I-labeled antifibrinogen antibodies were added to thrombin-stimulated platelets that were incubated with various unlabeled fibrinogen preparations for 5 and 60 minutes at 22°C. Antibody binding to platelets exposed to fibrinogen for 60 minutes is expressed as a percent compared with antibody binding to platelets exposed to fibrinogen for 5 minutes. Values represent mean ± SD, n = 3. † Kindly provided by Drs. C. Francis and P. J. Haidaris, University of Rochester School of Medicine, Rochester, New York. ‡ Kindly provided by Dr. B. Kudryk, New York Blood Center, New York, New York. § NR: not reactive; ND: not done.

Accessibility of Bound Fibrinogen to Plasmin The apparent correlation between platelet cytoskeletal activation and the stabilization of fibrinogen binding, together with the decreased accessibility of fibrinogen to antibody suggested that bound fibrinogen may be internalized. The observation that complete fibrinogen dissociation could be achieved in 30 minutes with 0.6 mU/ml plasmin, however, is inconsistent with this hypothesis. 20 ' 30 Closer examination of bound fibrinogen digestion by plasmin revealed that the rate of fibrinogen dissociation in response to low doses of plasmin (0.6 to 32 mU/ ml) decreased with time after platelet stimulation.30 Interestingly, chymotrypsin at 34 to 136 mU/ml failed to reveal statistically different rates of fibrinogen dissociation from platelets 5 and 60 minutes after fibrinogen binding,30 suggesting either specific changes in accessibility of plasmin binding or cleavage sites, or different

Downloaded by: University of British Columbia. Copyrighted material.

38

POST-FIBRINOGEN BINDING EVENTS—PEERSCHKE

Fibrinogen Expression on Fixed Platelets The previously described, progressive changes in accessibility of platelet-associated fibrinogen to antibody or plasmin were found to be considerably less marked if stimulated platelets were fixed with glutaraldehyde be­ fore fibrinogen binding was initiated.30 In contrast to fresh platelets, no differences in rates of bound fibrino­ gen degradation by plasmin were detected 5, 30, or 90 minutes after fibrinogen binding. Decreases in antifibrinogen antibody binding to fixed platelets were also attenu­ ated over the same time course. These data suggest that changes in the expression of bound fibrinogen by stimu­ lated platelets is an active process.

Effect of Fibrinogen Structure Because fibrinogen shares several RGD sequences on its alpha chains with other adhesive proteins capable of binding to the GPIIb-IIIa complex,34 the role of the fibrinogen carboxy-terminal alpha chain RGD sequence in the development of irreversible fibrinogen binding was investigated. The interaction of platelets with the fibrino­ gen degradation product 8D50 was examined.29 The Aa, Bβ, and gamma chain composition of 8D50 is summa­ rized in Table 3. Briefly, the predominant alpha chain remnant of this fibrinogen degradation product consisted of the amino-terminal third of the Aα chain (molecular weight, 25,000). The gamma chains of 8D50 remained intact, as assessed by analysis of Factor XIIIa crosslinking, and by the presence of only trace amounts of more anodal nonalpha chain bands on SDS-PAGE. No significant differences could be discerned between the amounts of intact fibrinogen and 8D50 that became irre­ versibly associated with platelets 5 and 60 minutes after platelet stimulation with either ADP or thrombin (Table 4). Furthermore, studies examining the modulation of platelet-associated fibrinogen expression using antibody probes revealed similar time-dependent decreases in ac­ cessibility of both a polyclonal antibody directed against

TABLE 3. Aα, Bβ, and Gamma Chain Composition of Native Fibrinogen, a Plasmic Fibrinogen Degradation Product, and a Naturally Occurring Fibrinogen Variant Mol Wt of Predominant Chain

(x10-3) Fibrinogen

Aα

Bβ

Gamma

Reference

Band I/gamma 50

70.9

54.5

50

29,38

8D50

25*

51.2†

50

29

gamma 5 7 5 /γ,

70.9,‡ 67.7†‡ 54.5

501‡, 57.5‡§ 36,38

* Reflects loss of carboxy-terminal residues including RGD572-574. † Reflects loss of amino-terminal residues. ‡ Denotes the presence of approximately equal amounts of these molecular weight species. § Denotes the presence of an additional carboxy-terminal amino acid sequence beginning four residues prior to the γ50 chain termination.

intact fibrinogen and a monoclonal antibody specific for an early fibrinogen fragment X (Table 2). To examine the contribution of the fibrinogen gamma chain dodecapeptide sequence to stabilizing platelet fibrinogen interactions, the binding of a plasma fibrinogen variant, designated gamma57.5 or γ' (Table 3) was examined.35-37 The increased molecular weight of gamma57.5 chains is due to an additional carboxy-termi­ nal amino acid sequence beginning four residues prior to the normal gamma chain termination.36 Purified fibrino­ gen gamma57.5 contained approximately equal amounts of normal gamma50 and elongated gamma57.5 chains (Ta­ ble 3). Platelet-bound fibrinogen gamma57.5 demon­ strated a similar gamma chain composition. The binding of the gamma57.5 fibrinogen variant became resistant to dissociation by EDTA or unlabeled fibrinogen over a similar time course and to the same extent as native fibrinogen binding in parallel studies (Table 4). Because direct interactions between stimulated platelets and gamma57.5 chains appear to be weak,38,39 the observed fibrinogen gamma57.5 binding and process­ ing may reflect the interaction of heterodimer fibrinogen molecules, containing one gamma50 and one gamma57.5

TABLE 4. Stabilization of Platelet-Fibrinogen Interactions: Comparison of Fibrinogen Variants and Degradation Products Fibrinogen

Irreversible Fibrinogen Binding* (%)

Reference

Gamma 50

5 2 + 15

38

Gamma 57.5

5 4 + 10

38

8D-50

5 7 + 11

29

* Values denote irreversible fibrinogen binding expressed as a percent relative to total fibrinogen binding.

Downloaded by: University of British Columbia. Copyrighted material.

susceptibilities of membrane glycoproteins, limiting the access of both antibodies and plasmin to bound fibrino­ gen. Concentrations of plasmin or chymotrypsin used for these experiments did not proteolyze GPIIb-IIIa, and only chymotrypsin caused a 33% decrease in platelet membrane GPIb.30 Analysis of bound fibrinogen degradation products resulting from plasmin or chymotrypsin cleavage by nonreduced SDS-PAGE were similar to degradation products obtained with native fibrinogen.30 These obser­ vations argue against the formation of covalent bonds between fibrinogen and activated platelet membrane con­ stituents.

39

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

TABLE 5. Effect of Monoclonal Antifibrinogen Antibodies on the Development of Irreversible Platelet-Fibrinogen Interactions Antibody* Designation

Specificity

Irreversible Fibrinogen Binding (%)†

Buffer control

—

39 ± 5

IC 2-2

A alpha

40 ± 6

1D4-1

A alpha 241-476

44 ± 6

18C-6

B beta 1-21

42 ± 8

FD4

Gamma Chain, Frag D

38 ± 3

* Monoclonal antibodies were a gift from Dr. B. Kudryk, New York Blood Center, New York, New York. †Thrombin-stimulated platelets were exposed to 125I-labeled fibrinogen that had been preincubated with buffer or monoclonal antibodies (fibrinogen: antibody, 1:10, mole:mole). Irreversible fibrinogen binding was assessed after 60 minutes, as described.29 Briefly, ethylene diaminetetraacetic acid (EDTA) (10 mM) was added to aliquots of individual platelet samples, and fibrinogen remaining bound was quantified 30 minutes later. Irreversible fibrinogen binding is expressed as fibrinogen remaining associated with platelets following exposure to EDTA relative to total fibrinogen binding. Values represent mean ± SD, n = 3.

chain, with platelets via the gamma50 chain or alpha chain RGD sequences. The intriguing possibility that irreversible fibrinogen binding may involve as yet undefined regions of the fibrinogen molecule is also under consideration. This hypothesis is supported by a recent report of fibrinogen binding to the leukocyte integrin Mac 1 via a non-RGDcontaining sequence in the E domain of the molecule.40 Thus far, studies have been performed with several monoclonal antibodies (kindly provided by Dr. B Kudryk, New York Blood Center, New York, NY) directed against epitopes on fibrinogen alpha, beta, and gamma chains to assess their effect on stabilizing platelet-fibrinogen interactions. These antibodies did not inhibit the initial interaction between fibrinogen and platelets, and could therefore be preincubated with fibrinogen and the combination added to stimulated platelets. As summarized in Table 5, however, none of the antibodies examined to date inhibited the formation of irreversible fibrinogen binding.

Requirement for GPIIb-IIIa Complexes Although fibrinogen binding to GPIIb-IIIa complexes is a prerequisite for the subsequent stabilization, 19,20,23 several observations suggest that intact GPIIb-IIIa complexes may not be required to mediate irreversible platelet-fibrinogen interactions. For example, exposure of stimulated platelets to EDTA at 37°C and at an alkaline pH, resulting in the dissociation of the GPIIb-IIIa complex, failed to dissociate irreversibly

bound fibrinogen.29 Disruption of GPIIb-IIIa complexes was quantified using the 10E5 monoclonal antibody41 and by two-dimensional crossed immunoelectrophoresis. The hypothesis that fibrinogen binding may involve either GPIIb or GPIIIa alone is further supported by observations of purified GPIIb and GPIIIa binding to immobilized fibrinogen.42 On whole platelets, such interactions may be facilitated by appropriate conformational changes in the receptor or its ligand subsequent to ligandreceptor interactions. Indeed, the exposure of neoantigenic determinants on both GPIIb and GPIIIa, referred to as ligand-induced binding sites (LIBS) has been described as a direct consequence of fibrinogen binding.43 Monoclonal antibodies recognizing two distinct LIBS on GPIIb and one on GPIIIa have been characterized.43,44

FUNCTIONAL IMPLICATIONS OF FIBRINOGEN BINDING AND PROCESSING The most significant post-fibrinogen binding event associated with platelet stimulation by a variety of agonists including thrombin is platelet aggregation.45 Several models have been proposed to explain the mechanism whereby fibrinogen supports platelet aggregation.12 Fibrinogen, by virtue of its symmetrical structure and multiple potential platelet binding sites, may form bridges between available receptors on adjacent platelets. It has been suggested that if fibrinogen were to bind to the platelet surface in a slanted or prone rather than upright orientation, several of the binding domains could act in concert to strengthen the binding of each molecule.12,46 The correlation between the extent of platelet aggregation and fibrinogen binding has been extensively documented (reviewed by Peerschke1). There are, however, exceptions to this relationship. Platelets chilled to 4°C bind fibrinogen but do not aggregate until they are rewarmed.47 Similarly, platelets fixed after stimulation with ADP bind fibrinogen but aggregate poorly.47 Moreover, two murine monoclonal antibodies, one directed against GPIIb (Tab) and one directed against GPIIIa (AP3), fail to inhibit fibrinogen binding, but inhibit ADP-induced platelet aggregation.48 In addition, platelets that have become refractory to stimulation with ADP, following an initial exposure to ADP in the presence of fibrinogen that did not result in their aggregation, fail to aggregate in response to a second stimulus of ADP despite sufficient membrane bound fibrinogen.49 These observations suggest that fibrinogen binding alone may not be sufficient to support platelet aggregation. It is likely that events occurring subsequent to fibrinogen binding to the platelet surface, such as conformational 33,43,44 or spatial50-52 receptor-ligand reorganization may also be involved. Investigations from several laboratories have shown that fibrinogen receptors on hu-

Downloaded by: University of British Columbia. Copyrighted material.

40

man platelets are mobile.50-52 GPIIb-IIIa are uniformly distributed over the surface of resting platelets, but clustering is observed after stimulation with thrombin. Clustering appears to be a function of ligand binding, since ADP-induced expression of fibrinogen receptors in the absence of exogenous fibrinogen fails to result in receptor redistribution, and platelet stimulation in the presence of fibrinogen, RGDS, or gamma401-411 supports this phenomenon.50 The purpose of receptor relocalization and clustering has not been determined, but it seems reasonable that the process may be important for platelet aggregation. Indeed, the selective distribution of fibrinogen clusters to regions of cell-cell contacts has been documented by electron microscopy.46 Recent studies using fibrinogengold as a probe further suggest that receptor ligand complexes may be translocated across the platelet surface membrane and cleared into channels of the open canalicular system.52 Conformational changes of bound fibrinogen and the occupied GPIIb-IIIa receptor complex reflect further post-fibrinogen binding events. The expression of RIBS33 on fibrinogen and LIBS43,44 on GPIIb-IIIa may impart unique functional properties to the ligand-receptor complex. Monoclonal antibodes recognizing two distinct LIBS on GPIIb, for example, have been shown preferentially to inhibit clot retraction and platelet adhesion to collagen, respectively.44 Moreover, a monoclonal antibody recognizing RIBS on platelet-associated fibrinogen was found to inhibit ADP-induced platelet aggregation.33 Other studies have revealed a temporal correlation between decreases in platelet-associated fibrinogen recognition by antibody probes and ADP- or thrombin-induced platelet aggregation.53 These observations support the hypothesis that qualitative changes in fibrinogen expression may modulate fibrin(ogen)-mediated support of platelet function.

SUMMARY AND HYPOTHESES The interaction between platelets and fibrinogen is a dynamic process that undergoes qualitative changes over time. Whereas fibrinogen binding to stimulated platelets is reversible initially, the interaction is progressively stabilized in a temperature- and agonist-dependent manner. Although the precise mechanism whereby this occurs is not understood, several conributing factors have been identified. Activation of the platelet cytoskeleton appears to be important for stabilizing fibrinogen binding, and release of alpha granule TSP, although not strictly required, may also contribute. The initial interaction between fibrinogen and the intact platelet GPIIb-IIIa complex is required, but thereafter the complex may not be necessary to sustain irreversible fibrinogen binding.

41 The observed processing of bound fibrinogen by stimulated platelets may have several functional implications. As previously suggested, stabilization of fibrinogen binding may contribute to the formation of stable interplatelet contacts supporting platelet aggregation and clot retraction. Processing of bound fibrinogen may initially enhance the exposure of epitopes involved in fibrinogen interaction with other ligands, such as TSP,27 fibronectin,54 Factor XIIIa,55 or plasmin.56 Further processing, associated with the decreased accessibility of bound fibrinogen to antibody probes and to plasmin may be consistent with previously observed cycling of the GPIIb-IIIa receptor pool57 and early phases involved in fibrinogen uptake or internalization,58,59 such as sequestration of fibrinogen in the surface canalicular system,52 which may not be accessible to antibody and only partially accessible to proteolytic enzymes. Alternatively, or in addition, the loss of fibrinogen accessibility to antibody probes may subserve a protective function by "removing" fibrinogen that does not participate in platelet aggregation from the platelet surface to limit the growth of a platelet plug or thrombus. Indeed recent evidence suggests that fibrinogen expression may be uniquely modulated.60 Using specific polyclonal and monoclonal antibodies, studies, comparing the RGD-dependent, GPIIb-IIIa mediated expression of fibronectin and von Willebrand factor on the surface of thrombin-stimulated platelets with that of fibrinogen as a function of time following ligand binding, demonstrated that the accessibility of platelet-associated fibronectin and von Willebrand factor to specific antibodies remained unchanged over a 90 minute time course, whereas the accessibility of bound fibrinogen to antibody decreased progressively. Further studies are required to understand better the biochemistry of "irreversible" platelet-fibrinogen interactions. The elucidation of fibrinogen structural requirements as well as platelet membrane constituents involved in stabilizing platelet fibrinogen interactions will be important. Careful examination of naturally occurring or synthetic fibrinogen mutants as well as further exploration of the agonist specificity involved in stabilization of platelet fibrinogen interactions may offer significant insights into the biochemistry and physiology of this phenomenon.

REFERENCES 1. Peerschke EIB: The platelet fibrinogen receptor. Semin Hematol 22:241-259,1985. 2. Kaplan KL, MJ Broekman, A Chernoff, GR Sesznik, M Drillings: Platelet alpha granule proteins: Studies on release and subcellular localization. Blood 53:604-618, 1979. 3. Hawiger J, S Parkinson, S Timmons: Prostacyclin inhibits mobili-

Downloaded by: University of British Columbia. Copyrighted material.

POST-FIBRINOGEN BINDING EVENTS—PEERSCHKE

4.

5. 6. 7.

8.

9.

10. 11.

12.

13.

14.

15.

16.

17.

18.

19.

20. 21.

22.

SEMINARS IN THROMBOSIS AND HEMOSTASIS—VOLUME 18, NO. 1, 1992 zation of fibrinogen binding sites on human ADP and thrombin treated platelets. Nature 283:195-197, 1980. Plow EF, GA Marguerie: Participation of ADP in the binding of fibrinogen to thrombin-stimulated platelets. Blood 56:553-555, 1980. Phillips DR, IF Charo, LV Parise, LA Fitzgerald: The platelet membrane glycoprotein IIb-IIIa complex. Blood 71:831-843, 1988. Hynes RO: Integrins: A family of cell surface receptors. Cell 48:549-554, 1987. Plow EF, JC Loftus, EG Levin, DS Fair, D Dixon, J Forsyth, MH Ginsberg: Immunologic relationship between platelet membrane glycoprotein GPIIb/IIIa and cell surface molecules expressed by a variety of cells. Proc Natl Acad Sci USA 83:6002-6006, 1986. 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. Coller BS: A new murine monoclonal antibody reports an activa­ tion-dependent change in the conformation and/or microenvironment of the platelet glycoprotein IIb-IIIa complex. J Clin Invest 76:101-108, 1985. Coller BS: Activation affects access to the platelet receptor for adhesive proteins. J Cell Biol 103:451-456, 1986. Hawiger J, M Kloczewiak, MA Bednarek, S Timmons: Platelet receptor recognition domains on the alpha chain of human fibrino­ gen: Structure function analysis. Biochemistry 28:2909-2914, 1989. Kloczewiak M, S Timmons, J Hawiger: Localization of a site interacting with human platelet receptors on the carboxyterminal segment of the human fibrinogen gamma chain. Biochem Biophys ResCommun 107:181-187, 1982. Santoro SA, WJ Lawing: Competition for related but nonidentical binding sites on the glycoprotein IIb-IIIa complex by peptides derived from platelet adhesive proteins. Cell 48:867-873, 1987. D'Souza SE, MH Ginsberg, TA Burke, SC-T Lam, EF Plow: Localization of an Arg-Gly-Asp recognition site within an integrin adhesion receptor. Science 242:91-93, 1988. D'Souza SE, MH Ginsberg, TA Burke, EF Plow: The ligand binding site of the platelet integrin GPIIb-IIIa is proximal to the second calcium binding domain of its alpha subunit. J Biol Chem 265:3440-3446, 1990. Lam SC-T, EF Plow, MA Smith, A Andrieux, J-J Ryclwaert, G Marguerie, MH Ginsberg: Evidence that arginyl-glycyl-aspartate peptides and fibrinogen gamma chain peptides share a common binding site on platelets. J Biol Chem 262:947-950, 1987. Bennett JS, SJ Shattil, JW Power, TK Gartner: Interaction of fibrinogen with its platelet receptor. Differential effects of alpha and gamma chain fibrinogen peptides on the glycoprotein IIb-IIIa complex. J Biol Chem 263:12948-12953, 1988. Parise LV, SL Helferson, B Steiner, L Nannizzi, DR Phillips: Synthetic peptides derived from fibrinogen and fibronectin change the conformation of purified platelet glycoprotein IIb-IIIa. J Biol Chem 262:12597-12602, 1987. Marguerie GA, TS Edgington, EF Plow: Interaction of fibrinogen with its platelet receptor as part of a multistep reaction in ADPinduced platelet aggregation. J Biol Chem 255:154-161, 1980. Peerschke EIB, JA Wainer: Examination of irreversible plateletfibrinogen interactions. Am J Physiol 248:C466-C472, 1985. Peerschke EIB: Time dependent association between platelet bound fibrinogen and the Triton X-100 insoluble cytoskeleton. Blood 77:508-514, 1991. Marguerie GA, N Thomas-Maison, M-J Larrieu, EF Plow: The interaction of fibrinogen with human platelets in a plasma milieu. Blood 59:91-95, 1982.

23. Kornecki E, S Niewiarowski, TA Morinelli, M Kloczewiak: Effect of chymotrypsin and adenosine diphosphate on the exposure of fibrinogen receptors on normal human and Glanzmann's thrombas­ thenia platelets. J Biol Chem 256:5696-5701, 1981. 24. Casella JF, NC Masiello, S Lin, W Bell, MB Zucker: Identifica­ tion of fibrinogen derivatives in the Triton-insoluble residue of human blood platelets. Cell Motil 3:21-30, 1982. 25. Laudano AP, RF Doolittle: Synthetic peptide derivatives that bind to fibrinogen and prevent the polymerization of fibrin monomers. Proc Natl Acad Sci USA 75:3085-3089, 1978. 26. Fox JEB, DR Phillips: Polymerization and organization of actin filaments with platelets. Semin Hematol 20:243-281, 1983. 27. Asch AS, RL Nachman: Thrombospondin: Phenomenology to function. Prog Hemost Thromb 9:157-175, 1989. 28. Leung LLK: Role of thrombospondin in platelet aggregation. J Clin Invest 74:1764-1772, 1984. 29. Peerschke EIB: Irreversible platelet-fibrinogen interactions occur independently of fibrinogen alpha chain degradation and are not mediated by intact platelet membrane glycoprotein IIb-IIIa com­ plexes. J Lab Clin Med 111:84-92, 1988. 30. Peerschke EIB: Decreased accessibility of platelet-bound fibrino­ gen to antibody and enzyme probes. Blood 74:682-689, 1989. 31. Francis CW, RL Nachman, VJ Marder: Plasma and platelet fibrin­ ogen differ in gamma chain content. Thromb Haemost 51:84-88, 1984. 32. Mosesson MW, GA Homandberg, DL Amrani: Human platelet fibrinogen gamma chain structure. Blood 63:990-995, 1984. 33. Zamarron C, MH Ginsberg, EF Plow: Receptor induced binding sites (RIBS) are exposed in fibrinogen as a consequence of its interaction with platelets. Blood 74:208a, 1989. 34. Ruoslahti E, MD Pierschbacher: Arg-Gly-Asp: A versatile cell recognition signal. Cell 44:517-518, 1986. 35. Francis CW, VJ Marder, SE Martin: Demonstration of a large molecular weight variant of the gamma chain of normal human plasma fibrinogen. J Biol Chem 255:5599-5604, 1980. 36. Wolfenstein-Todel C, MW Mosesson: Human plasma fibrinogen heterogeneity: Evidence for an extended carboxy-terminal se­ quence of a normal gamma chain variant (γ'). Proc Natl Acad Sci USA 77:5069-5073, 1980. 37. Peerschke EIB, CW Francis, VJ Marder: Fibrinogen binding to human blood platelets: Effect of gamma chain carboxyterminal structure and length. Blood 67:385-390, 1986. 38. Haidaris PJ, EIB Peerschke, VJ Marder, CW Francis: The C-terminal of the gamma 57.5 chain of human fibrinogen constitutes a plasmin sensitive epitope that is exposed in crosslinked fibrin. Blood 74:2437-2444, 1989. 39. Kirschbaum N, MW Mosesson, D Amrani: Assessment of plateletfibrinogen interactions using a native, monovalent fibrinogen frag­ ment D. (Abst.) Circulation 82:III-132, 1990. 40. Altieri DC, FR Agbanyo, J Plescia, MH Ginsberg, TS Edgington, EF Plow: A unique recognition site mediates the interaction of fibrinogen with the leukocyte integrin Mac 1. J Biol Chem 265:12119-12122, 1990. 41. Coller BS, EI Peerschke, LE Scudder, CA Sullivan: A murine monoclonal antibody that completely blocks the binding of fibrin­ ogen to platelets produces a thrombasthenic-like state in normal platelets and binds to glycoproteins IIb and/or IIIa. J Clin Invest 72:325-338, 1983. 42. Parise LV, B Steiner, L Nannizzi, DR Phillips: Fibrinogen binding sites exist on platelet glycoproteins IIb and IIIa. Blood 70:357a, 1987. 43. Frelinger AL 3d, SC-T Lam, EF Plow, MA Smith, JC Loftus, MH Ginsberg: Occupancy of an adhesive glycoprotein receptor modu-

Downloaded by: University of British Columbia. Copyrighted material.

42

44.

45.

46.

47.

48.

49.

50.

51.

lates expression of an antigenic site involved in cell adhesion. J Biol Chem 263:12397-12402, 1988. Frelinger AL 3d, I Cohen, EF Plow, MA Smith, J Roberts, SCT Lam, MH Ginsberg: Selective inhibition of integrin function by antibodies specific for ligand-occupied receptor conformers. J Biol Chem 6346-6352, 1990. Peerschke EI, MB Zucker, RA Grant, MJ Johnson, JJ Egan: Correlation between fibrinogen binding to human platelets and platelet aggregability. Blood 55:841-847, 1980. Moon DG, JR Shainoff, SR Gonda: Electron microscopy of platelet interaction with heme-octapeptide labeled fibrinogen. Am J Physiol 259:C611-C618, 1990. Peerschke EI, MB Zucker: Fibrinogen receptor exposure and aggregation of human blood platelets produced by ADP and chilling. Blood 57:663-669, 1981. Newman PJ, RP McEver, MP Doers, TJ Kunicki: Synergistic action of two murine monoclonal antibodies that inhibit ADPinduced platelet aggregation without blocking fibrinogen binding. Blood 69:668-676, 1987. Peerschke EIB: Ca +2 mobilization and fibrinogen binding of platelets refractory to adenosine diphosphate stimulation. J Lab Clin Med 106:111-122, 1985. Isenberg WM, RP McEver, DR Phillips, MA Shuman, DF Bainton: The platelet fibrinogen receptor: An immunogold-surface replica study of agonist-induced ligand binding and receptor clustering. J Cell Biol 104:1655-1663, 1987. Loftus JC, RM Albrecht: Use of colloidal gold to examine fibrinogen binding to human platelets. Scanning Electron Microsc 4:1995-1999, 1983.

43 52. Leistikow EA, MI Barnhart, G Escolar, JG White: Receptor-ligand complexes are cleared to the open canalicular system of surface activated platelets. Br J Haematol 74:93-100, 1990. 53. Peerschke EIB: Modulation of fibrinogen expression on stimulated platelets: Correlation with refractoriness. (Abst.) Thromb Haemost: 1991. (in press) 54. Plow EF, GA Marguerie, MH Ginsberg: Fibronectin binding to thrombin stimulated platelets: Evidence for fibrin(ogen) dependent and independent pathways. Blood 66:26-32, 1985. 55. Chen R, RF Doolittle: Gamma-gamma crosslinking sites in human and bovine fibrin. Biochemistry 10:4486-4491, 1971. 56. Lucas MA, LJ Fretto, PA McKee: The relationship of fibrinogen structure to plasminogen activation and plasmin activity during fibrinolysis. Ann NY Acad Sci 408:71-91, 1984. 57. Wencel-Drake JD: Plasma membrane GPIIb/IIIa. Evidence for a cycling receptor pool. Am J Pathol 136:61-70, 1990. 58. Harrison P, B Wilbourn, N Debili, W Vainchenker W, J BretonGorius, AS Lawrie, J-M Masse, GF Savidge, EM Cramer: Uptake of plasma fibrinogen into the alpha granules of human megakaryocytes and platelets. J Clin Invest 84:1320-1324, 1989. 59. Handagama PJ, MA Shuman, DF Bainton: Incorporation of intravenously injected albumin, immunoglobulin G, and fibrinogen in guinea pig megakaryocyte granules. J Clin Invest 84:73-82, 1989. 60. Peerschke EIB. Selective modulation of fibrinogen expression on thrombin-stimulated platelets. (Abst.) Circulation 82:III-368, 1990.

Downloaded by: University of British Columbia. Copyrighted material.

POST-FIBRINOGEN BINDING EVENTS—PEERSCHKE