CLINICAL PATHOLOGY Review Article

Platelet Membrane Glycoproteins Functional Characterization and Clinical Applications ELLINOR I. B. PEERSCHKE, PH.D.

Adhesion Receptors of the Integrin Family

Integrins are alpha/beta heterodimer protein complexes involved in cell-matrix and/or cell-cell interactions (reviewed by Phillips and others,6 Bennett,10 and Ginsberg and others''). They are distributed widely on the surfaces of nearly all adherent cells from mammals to species of invertebrate origin.12"14 Each heterodimer receptor complex in the superfamily is composed of a unique alpha PLATELET MEMBRANE GLYCOPROTEIN subunit and one of three different beta subunits, desigSTRUCTURE AND FUNCTION nated betai, beta2, and beta3. The beta, subunit is shared Many of the receptors involved in platelet adhesion to by receptors first described on activated T lymphocytes extracellular matrix components in the blood vessel wall, several weeks after in vitro stimulation with antigen or mitogen, and thus termed very late antigens (VLA) (reviewed in Hemler" and Ruoslahti and Giancotti16). This From the Department of Pathology, SUNY at Stony Brook. Stony group includes platelet membrane receptors for fibronecBrook, New York. tin (GPIc/IIa), collagen (GPIa/IIa), and laminin (Ic*/Ha). Supported in part by grant HL28183 from the National Heart, Lung Integrins possessing the beta2 subunit are found predomand Blood Institute, and grant 900994 from the American Heart Assoinantly on leukocytes.17 Receptors associated with the ciation-Wyeth Ayers Laboratories. Received October 31, 1991; revised manuscript accepted for publibeta3 subunit constitute the cytoadhesin subfamily, which cation January 10, 1992. includes platelet membrane receptors for fibrinogen Address reprint requests to Dr. Ellinor Peerschke: University Hospital, (GPIIb/IIIa) and vitronectin.6'9"" In general, integrins L-3, SUNY at Stony Brook, Stony Brook, New York 11794-7300.

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platelet aggregation, and platelet interaction with other cells have been identified, cloned, and sequenced (reviewed by Bennett910). This information has allowed their classification within a number of gene families, including the integrin gene family, the leucine-rich glycoprotein gene family, and the selectin gene family, composed of receptors sharing structural and functional similarities and found on a variety of cell types (reviewed in Phillips and others,6 Bennett,10 and Ginsberg and others''). Other surface membrane glycoproteins participating in hemostasis and belonging to the immunoglobulin gene superfamily have been identified, as well as platelet proteins that appear unrelated to any known gene family. A summary of platelet membrane glycoprotein classification, together with salient structural and functional characteristics of major glycoproteins, is presented in Table 1.

The role of platelets in hemostasis and thrombosis is well established.' Platelet plug formation at sites of vascular injury is responsible for the primary arrest of bleeding and is vital to the maintenance of vascular integrity. Under pathologic conditions, however, uncontrolled thrombosis can lead to major tissue damage as a result of vessel occlusion or thrombus embolization. Platelet plug formation in hemostasis and thrombosis is a complex process, mediated in large part by the interaction of platelet membrane glycoproteins (GP) with components of the vessel wall and/or plasma (for review, see Fitzgerald and Phillips,2 Coller and others,3 George and associates,4 and Kieffer and Phillips5). Considerable progress has been made during the last decade in identifying and characterizing platelet membrane components involved in platelet-vessel wall and platelet-platelet interactions (reviewed in Phillips and others,6 Tomasini and Mosher,7 and Coller8). It is the aim of this review to provide an overview of the structure and function of major platelet membrane glycoproteins and to highlight examples of the direct application of emerging concepts, defining the molecular interactions of platelets with their environment, to the diagnosis of platelet defects and the development of new therapies for thrombotic disorders.

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Review Article TABLE 1 . PLATELET MEMBRANE GLYCOPROTEINS: OVERVIEW OF BIOCHEMICAL AND FUNCTIONAL CHARACTERISTICS

Glycoprotein

Alternate Designation

Molecular Weight (reduced)*

Gene Familyf

167,000/130,000 135,000,27,000/130,000 125,000/130,000 125,000,23,000/110,000

Integrin Integrin Integrin Integrin

Vitronectin receptor

135,000,25,000/110,000

Integrin (B3)

GP Ib/IX GPV

145,000, 24,000/17,000 82,000

LRG LRG

GP GP GP GP

la/Ila Ic/IIa Ic*/IIa Ilb/IIIa

VLA-2 VLA-5 VLA-6

GPIV

GPIIIb, CD36

88,000

GMP 140

PADGEM, CD62

140,000

(B|) (B|) (B0 (B3)

Ligand% Collagen Fn Laminin Fb, vWF Vn, Fn Vn, ?vWF ?Fn vWF thrombin ?

?

TSP Collagen

Selectin

9

Function

Expression%

Adhesion Adhesion Adhesion Aggregation Adhesion Adhesion

C C C C,S

Adhesion Thrombin substrate Adhesion

C C

Platelet-leukocyte interactions

C

C S

X Fb = fibrinogen; Fn = hbronectin; Vn = vitronectin; TSP = thrombospondin; vWF = von Willebrand factor. § C = constitutive expression; S = expression or change in expression after platelet stimulation. [| B = beta subunits.

mediate cell adhesion by interacting with a variety of extracellular glycoprotein ligands containing the amino acid sequence Arg-Gly-Asp (RGD)." 1 2 1 8 Platelet Cytoadhesins. The GPHb-HIa Fibrinogen Receptor. The glycoprotein Hb-IIIa complex is probably the most abundant integrin on the platelet membrane. 619 It exists as a calcium-dependent, heterodimer complex20 that contains the antigens responsible for most cases of post-transfusion purpura and neonatal thrombocytopenia.21 GPIIb expresses the Bak(Lek) antigen,22 whereas GPIIIa exhibits the PI A 2 3 and Pen(YUK) antigens.24 Glycoprotein lib has a molecular weight of 136,000 and is composed of a larger alpha (125,000) and a smaller beta (23,000) subunit, linked by a single disulfide bridge. Glycoprotein Ilia, or beta3, is a disulfide bond-rich, single chain protein with an apparent molecular weight of 90,000 on nonreduced sodium dodecyl sulfate gels, and an apparent molecular weight of 110,000 after disulfide bond reduction (reviewed in Kieffer and Phillips,5 Phillips and associates,6 and Bennett9). Normal platelets contain at least 50,000 molecules of GPIIb-IIIa,25 representing 1% to 2% of the total platelet protein. Additional complexes are present in membranes of the platelet surface canalicular system and alpha granules.26,27 These can be expressed at the surface after platelet activation.28

of the proximity of the GPIIb and GPIIIa genes, both are located on the proximal portion of the long arm of chromosome 17 at q21-23 (reviewed by Bennet 910 ), thrombasthenia may arise as a consequence of defective coordinate control of GPIIb and GPIIIa gene expression. Alternatively, studies of GPIIb and GPIIIa biosynthesis predict that thrombasthenia may also result from defects in the synthesis of either subunit alone because membrane expression of the GPIIb/IIIa complex appears to require heterodimer complex assembly in the endoplasmic reticulum. 910 Indeed, the molecular defect, in the limited number of patients studied, has been localized to either the GPIIb or GPIIIa gene. 3132 Although GPIIb and GPIIIa are constitutively expressed on resting platelets, the heterodimer complex binds soluble adhesive proteins (fibrinogen, von Willebrand factor [vWF], or fibronectin) only after platelet activation (reviewed in Coller8). Recognition of ligands by the GPIIbIIIa heterodimer complex involves the RGD sequence33 and requires either calcium or magnesium.25 Peptides corresponding to the 15 amino acids of the carboxyterminus of the fibrinogen gamma chain, however, also completely inhibit fibrinogen interactions with stimulated platelets,34 and abrogate the binding of vWF and fibronectin to the GPIIb-IIIa complex.35 Current evidence36"38 suggests that the RGD and gamma chain binding sites in GPIIb/IIIa are spatially distinct, and supports the hypothesis that the binding of one peptide induces a conformational change in the GPIIb-IIIa complex that excludes binding of the other. Platelet aggregation under normal circumstances is overwhelmingly mediated by the interaction of fibrinogen with the GPIIb-IIIa receptor.39 In the absence of plasma

The presence of surface membrane GPIIb-IIIa is an absolute requirement for platelet aggregation (reviewed in Nurden and Caen 29 ). Lack of functional GPIIb/IIIa complexes is responsible for the congenital bleeding disorder called Glanzmann's thrombasthenia. Glanzmann's thrombasthenia is an autosomal recessive hemorrhagic disorder resulting, in most cases, from a parallel decrease in the platelet content of GPIIb and GPIIIa. 2930 Because

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• Numbers separated by commas designate the molecular weight of reduced subunits; numbers separated by slashes indicate the molecular weight of alpha and beta subunits, respectively, t LRG = leucine-rich glycoprotein family.

PEERSCHKE Platelet Glycoproteins

is difficult to distinguish from GPIc on one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).58 However, the two proteins can be separated readily by two-dimensional isoelectric focusing/ SDS PAGE and by peptide mapping. Magnesium, but not calcium, supports laminin binding to the VLA-6 integrin. 57 The Leucine-Rich Glycoproteins The leucine-rich glycoprotein gene family represents a relatively new family of proteins that share a common structural motif, composed of a leucine-rich, 24-aminoacid consensus sequence.59 This family includes a variety of proteins with no apparent functional similarities. Both the platelet membrane GPIb/IX complex and GPV are members of the leucine-rich glycoprotein family. GPIb/IX: The von Willebrand Factor Receptor. GPIb is the most abundant sialoglycoprotein on the platelet membrane, with approximately 25,000 molecules/platelet (reviewed in Clemetson60). It consists of two disulfidelinked subunits, GPIb alpha (molecular weight 145,000) and GPIb beta (molecular weight 24,000), both of which are transmembrane proteins. The alpha and beta chains are encoded by two distinct genes. The gene for GPIb alpha has been located on chromosome 17, and the gene for GPIb beta on chromosome 22 (reviewed in Bennett9 and Wenger and associates61). GPIb is tightly but noncovalently complexed with GPIX in a 1:1 heterodimer complex.62 GPIb alpha is susceptible to proteolysis by a number of enzymes63 that release the extracellular domain called glycocalicin. GPIb alpha also has been reported to bind thrombin 64 and is the site of interaction of quinine/quinidine drug-dependent antibodies.65 Its primary function, however, is in mediating platelet adhesion to vascular subendothelium through immobilized vWF (reviewed in George and colleagues4). Platelets from patients with the Bernard-Soulier syndrome fail to bind vWF and lack GPIb/IX. 29 Furthermore, monoclonal antibodies directed against GPIb alpha can induce a Bernard-Soulier-like phenotype in vitro.66 Glycoprotein V. Glycoprotein V is an 82,000-molecularweight platelet membrane glycoprotein (reviewed in Fitzgerald and Phillips,2 George and colleagues,4 and KiefFer and Phillips5). This glycoprotein also appears to be associated with the GPIb/IX complex and is deficient in patients with the Bernard-Soulier syndrome (reviewed in George and colleagues4). Although the function of GPV is unknown, it is the only major platelet membrane glycoprotein that is hydrolyzed by thrombin during platelet stimulation.67 The possible function of GPV hydrolysis

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fibrinogen, however, such as in patients with afibrinogenemia, vWF has been shown to substitute for fibrinogen.40 The vitronectin receptor. In comparison with GPIIb/ Ilia, the vitronectin receptor represents a minor integrin on platelets.41 Its alpha subunit (a v ) has 35% sequence identity with GPIIb and forms a calcium-independent complex with beta subunits that are identical to GPIIIa. The vitronectin receptor binds several RGD-containing adhesive proteins such as vitronectin, fibronectin, vWF,42 and thrombospondin. 43 Its function is constitutive and does not require platelet activation. Interestingly, patients with thromboasthenia have been described whose platelets lack GPIIb/IIIa complexes but express normal to increased amounts of vitronectin receptors.44 Because both receptors share the same beta subunit (B3), this observation implicates defective synthesis of GPIIb in the expression of the thromboasthenic phenotype in these patients. The VIA Family of Receptors. Three Betai Integrins Have Been Identified on Platelets. These function primarily as receptors for collagen, fibronectin, and laminin. The betai subunit corresponds to GPIIa, which has an apparent molecular weight of 130,00045 and bears the Br3/ Brb platelet alloantigen system.46'47 The Collagen Receptor. The VLA-2 platelet collagen receptor (GPIa/IIa) is present at about 2,000 copies per platelet.48'49 Its alpha subunit, GPIa, has an unreduced molecular weight of 153,000 that increases to 167,000 after reduction of disulfide bonds. 50 This receptor functions not only as an adhesive protein receptor, but also as an agonist receptor. Its role in collagen-induced platelet aggregation was first suggested when a patient whose platelets failed to respond to collagen were found to lack GPIa. 5 ' Although a variety of collagen-binding proteins have been described on platelets, GPIa/IIa seems to play a primary role in platelet-collagen interactions (reviewed in Santoro52). The Fibronectin Receptor. The fibronectin receptor (VLA-5) is another minor integrin on platelet membranes. It is composed of a heterodimer complex of GPIc and GPIIa.53,54 This receptor interacts with fibronectin and laminin and does not require platelet activation for activity. Indeed, platelets from patients with Glanzmann's thromboasthenia, lacking GPIIb/IIIa, spread on fibronectin-coated surfaces under both static and flow conditions55 via the VLA-5 fibronectin receptor. Adhesion is mediated by recognition of RGD-containing peptides in the ligand. The Laminin Receptor. Platelets also adhere to but do not spread on laminin-coated surfaces.56 The role of VLA6 in platelet interactions with laminin was established in studies using a monoclonal antibody against VLA-6.57 The alpha subunit of VLA-6, sometimes designated Ic*,

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in thrombin-induced platelet activation remains controversial, however.68

TABLE 2. PLATELET MEMBRANE GLYCOPROTEINS: CLINICAL APPLICATIONS Application

The Selectins: GMP 140

Other Platelet Proteins Involved in Hemostasis and Thrombosis GPIV, also known as GPIIIb or CD36, is an 88,000molecular-weight, major platelet glycoprotein that appears unrelated to any known gene family. It is highly glycosylated, expresses the OKM 5 antigen, and is resistant to proteases when expressed in the platelet membrane. 73 ' 74 In addition to cells of megakaryocyte lineage, GPIV has also been identified on melanoma cells, monocytes, and endothelial cells.75 GPIV on platelets functions as a receptor for the alpha granule protein thrombospondin. 76 It also binds to Type I collagen fibrils, and antibodies raised against GPIV inhibit collagen-induced platelet aggregation.77 Interestingly, GPIV on endothelial cells mediates the binding of Plasmodium falciparum-infected erythrocytes (reviewed in Howard and Gilladoga78), an activity that appears to be separate from the receptor's ability to bind thrombospondin. 79 CLINICAL APPLICATIONS: DIAGNOSTIC AND THERAPEUTIC RELEVANCE OF PLATELET MEMBRANE GLYCOPROTEINS Our understanding of platelet membrane glycoprotein structure and function has been derived in large part as a result of the development and characterization of many monoclonal antibodies. Some of these antibodies have been useful in the diagnosis of bleeding and thrombotic disorders. The definition of platelet-adhesive interactions at the molecular level has also prompted the design of new therapeutic modalities based on the inhibition of platelet integrins with monoclonal antibodies or synthetic and naturally occurring peptides that mimic appropriate

A.J.C.P. •

Diagnostic Platelet function defects Thrombocytopenia Detection of in vivo platelet activation Thrombus imaging Antithrombotic therapy

Agents

GP lb, GP Ilb/IIla

Monoclonal antibodies

GP lb GP Hb/IIIa, GMP 140

Monoclonal antibodies Monoclonal antibodies

GP Ilb/IIla, GMP 140 GP Ilb/IIla

Monoclonal antibodies Monoclonal antibodies, synthetic peptides, snake venoms

ligand recognition sequences. A summary of applications of monoclonal antibodies and synthetic peptides to diagnostic and therapeutic regimens is presented in Table 2. Although inherited and acquired platelet-related bleeding disorders are relatively rare, thrombosis is recognized as the principal cause for cardiovascular morbidity and mortality (reviewed in John and others80). Because vascular injury and thrombus formation represent key events in the pathogenesis of vascular disease, including atherosclerosis, the development of specific platelet inhibitors may offer an important adjunct to antithrombotic therapy. Diagnostic Approaches Recent diagnostic approaches have taken advantage of the inherent specificity and selectivity of monoclonal antibodies directed against various platelet membrane glycoproteins. The first applications of monoclonal antibodies were directed toward the rapid diagnosis of congenital platelet dysfunctions involving GPIIb/IIIa and GPIb. Diagnosis of Qualitative Platelet Disorders. Although the diagnosis of thrombasthenia and the Bernard-Soulier syndrome can be made by traditional platelet function analysis, including, at a minimum, evaluation of the bleeding time, platelet retention, and platelet aggregation studies, these assays are time consuming and require large volumes of blood and considerable manipulation before analysis. In addition, the performance of functional studies on platelets from patients with the Bernard-Soulier syndrome is often complicated because these platelets are generally larger in size81 and more difficult to separate from blood. Thus, platelet glycoprotein analysis, using monoclonal antibodies, provides an alternative, rapid, diagnostic procedure for the detection of functional defects caused by the altered expression of key platelet surface components. A whole blood assay for the diagnosis of the BernardSoulier syndrome and thrombasthenia has been described

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The selectins comprise a family of cell-surface receptors that possess an amino terminal lectin-like domain, an adjacent epidermal growth factor-like domain, and multiple short consensus repeat units homologous to the complement regulatory proteins.69 GMP 140, also called PADGEM or CD62, is a 140,000-molecular-weight platelet constituent residing in alpha granule membranes. 70 It is expressed on the platelet surface only after platelet activation and alpha granule secretion.70*71 GMP 140 has been shown to mediate the adherence of neutrophils and monocytes to activated platelets in a calcium-dependent manner. 72

Target Glycoprotein

PEERSCHKE Platelet Glycoproteins

Detection of Circulating Activated Platelets. Because platelet activation accompanies a number of vascular disorders, such as unstable angina, peripheral vascular disease, stroke, angioplasty, and coronary thrombolysis,89"92 considerable effort has been exerted during the last two decades to develop more sensitive and specific methods to detect activated, circulating platelets. The most reliable markers of in vivo platelet activation consist of substances, released from platelets after activation, measured in the plasma or urine: platelet factor 4 (PF4), /3-thromboglobulin (/3-TG), and metabolites of thromboxane A2 (reviewed in Gershlick93). These markers have not achieved widespread clinical acceptance, however, because of technical limitations pertaining to sample collection, processing, and analysis. Several changes in surface membrane glycoprotein expression can be detected during platelet activation with specific murine monoclonal antibodies. For example, changes in the conversion of the GPIIb-IIIa complex to a functional fibrinogen receptor can be detected, 9495 and platelet activation with accompanying alpha granule release can be ascertained by examining GMP-140/PADGEM expression.96 Thus, assays have been designed that combine the use of activation-specific monoclonal antibodies with the sensitivity of flow cytometry.94"96 These are performed on whole blood, require only microliter volumes of sample, and facilitate the detection of platelet subpopulations that are heterogeneous with respect to their activation status. In Vivo and In Vitro Thrombus Imaging. New techniques in thrombus imaging, using monoclonal antibodies directed against GPIIb/IIIa 97 and GMP 140,98 have been evaluated in primates. These may offer several advantages over the use of'" ' indium-labeled platelets. For example, because ' " ' indium labeling of platelets is performed ex vivo, the potential for ex vivo platelet activation is unavoidable. Furthermore, using ' " ' indium-labeled platelets for thrombus imaging usually results in the incorporation of only small amounts of radioactive tracer in the thrombus, with relatively large amounts of tracer remaining in the circulation, generating a small signal-tonoise ratio. This allows only for the detection of large thrombi with ease and reliability. Because labeled monoclonal antibodies can be injected directly, problems associated with ex vivo labeling are circumvented. Furthermore, activation-dependent monoclonal antibodies, such as those recognizing GMP-140/PADGEM, 98 may prove to be particularly useful because the target antigen does not circulate in blood (resting platelets are unreactive). Unbound label appears to be cleared rapidly, leading to low background activity and concentration of radioactivity at the site of the thrombus.

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using monoclonal antibodies directed against GPIb and GPIIb/IIIa, respectively.82,83 The discovery of specific glycoprotein deficiencies associated with congenital bleeding disorders, such as thromboasthenia and the Bernard-Soulier syndrome, also has led to the development of methods to identify carriers based on partial glycoprotein deficiencies.84 Such testing is especially valuable because these disorders are transmitted in an autosomal recessive manner and heterozygous carriers are often free of both clinical symptoms and laboratory abnormalities, as assessed by traditional platelet function studies. Furthermore, requests by families who already have children affected by these disorders have prompted the development of microtechniques using polyclonal and monoclonal antibodies for antenatal diagnosis.8586 As the panel of monoclonal antibodies directed against GPIIb/IIIa has expanded, antibodies emerged that distinguish between resting, activated, and ligand-occupied forms of the receptor. Combined with recent advances in flow cytometry, these antibodies have made it possible to evaluate the function of platelet fibrinogen receptors,87 and thus to distinguish between quantitative and qualitative defects in GPIIb/IIIa. These techniques also have been applied to assess GPIIb/IIIa receptor status in patients with myeloproliferative syndrome and other suspected, acquired aggregation disorders.87 Diagnosis of Quantitative Platelet Disorders. In addition to aiding in the diagnosis of qualitative platelet defects, certain monoclonal antibodies have been useful in the evaluation of quantitative platelet disorders. Because it is sometimes difficult to determine whether thrombocytopenia is caused by underproduction of platelets (decreased bone marrow megakaryocytes) or an increased rate of platelet destruction (normal to increased bone marrow megakaryocytes), a test examining plasma glycocalicin concentrations using a monoclonal antibody directed against GPIb alpha may serve as a useful, noninvasive adjunct in classifying thrombocytopenia.88 As described in above, glycocalicin is a carbohydraterich hydrophobic fragment of GPIb that is released from the platelet membrane by a variety of proteases. The results of a small study of 33 patients suggest that plasma glycocalicin concentrations may reflect overall platelet turnover, platelet mass, and rate of platelet destruction.88 These findings are encouraging, particularly as the evaluation of patients with thrombocytopenia usually includes bone marrow analysis and platelet survival studies, both of which have serious drawbacks. Bone marrow aspiration and biopsy are invasive procedures and are subject to sampling errors and subjective interpretation, whereas platelet life-span measurements expose the patient to radiation and are complex to interpret.

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Antithrombotic

Therapy

Monoclonal Antibodies as Antithrombotic Agents. Studies using various animal models have shown that inhibition of platelet aggregation with potent anti GPIIb/ Ilia monoclonal antibodies prevents platelet deposition on vascular grafts, overcomes resistance of platelet-rich thrombi to thrombolysis, and accelerates and sustains coronary artery reperfusion with decreasing doses of recombinant-tissue plasminogen activator (rt-PA) (reviewed in Becker and Gore," Coller and associates,100 and Ruggeri101'102). Recently, phase I clinical trials were initiated using F(ab')2 fragments of one anti-GPIIb-IIIa monoclonal antibody, 7E3, in patients with unstable angina (reviewed in Coller and associates100). After administration of 7E3, none of the patients experienced symptomatic angina while their bleeding times were significantly prolonged. Some patierts had return of pain when their bleeding time returned to normal, even though platelet aggregation remained significantly inhibited. These initial results are very encouraging and suggest that platelets play an important role in the pathogenesis of unstable angina. One of the anticipated risks associated with monoclonal antibody-induced inhibition of platelet aggregation, however, is bleeding. 10° The data from the phase I study using 7E3 indicate that after a single dose of antibody, causing near-quantitative receptor blockade and marked prolongation of the bleeding time, the return of a normal bleeding time occurred during a period of 12 to 24 hours. Some inhibition of platelet aggregation persisted for at least 3 days. Because the concentration of free antibody de-

CONCLUDING REMARKS AND FUTURE TRENDS The past decade has witnessed a virtual explosion of information regarding the structure and function of

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Platelets have been implicated in a variety of vaso-occlusive and thromoembolic disorders that together constitute the most common cause of death in the United States. Although some studies with available anti-platelet agents have been encouraging, in general these drugs have shown only modest clinical efficacy (reviewed by John and others80). Alternative strategies have been developed using monoclonal antibodies or synthetic peptides to inhibit platelet function. The interaction of platelets with fibrinogen has been a primary target for these approaches, as it appears to be the final common pathway in plateletplatelet interactions. Furthermore, platelet-mediated thrombosis is a significant pathogenic mechanism underlying the limited efficacy of thrombolytic therapy, and currently available thrombolytic agents suffer from limitations, including resistance to reperfusion, acute coronary reocclusion, prolonged time to restoration of antegrade coronary flow, and bleeding tendency (reviewed in Becker and Gore"). Thus, the development of more potent anti-platelet agents in this setting is also a primary goal.

creased, in less than 1 hour after administration, however, to levels too low to produce significant inhibition of fresh platelets, platelet transfusion should readily reverse any hemorrhagic diathesis produced by the antibody. Synthetic Peptides and Snake Venoms as Antithrombotic Agents. In addition to monoclonal antibodies, GPIIb/IIIa function can be blocked by small synthetic molecules containing the RGD sequence (reviewed in Ruggeri101102). The low affinity of most of the compounds studied so far, however, presents a limitation to their possible pharmacologic use. It is possible, however, to tailor molecules with the characteristic specificity and affinity necessary for pharmacologic inhibition of GPIIb/IIIa. A family of snake venoms derived from pit vipers of the genera Trimeresurus contain RGD recognition sequences and appear to be very potent inhibitors of GPIIb/ Ilia (reviewed in Dennis and associates103). One such venom, kistrin, a 68-amino-acid polypeptide from the venom of the Malayan pit viper Agkistrodon rhodostoma, was shown to be an effective, rapidly reversible inhibitor ofGPIIb-IHa in rabbits.103 The efficacy of kistrin in thrombolysis associated with the administration of rt-PA also was evaluated recently in a canine model of coronary artery thrombosis. 104 Kistrin increased the rate and extent of thrombolysis with a reduced dose of rt-PA and prevented reocclusion. At an effective dose, it was associated with only a transient prolongation of the bleeding time and inhibition of platelet aggregation. Although preliminary results of fibrinogen receptor blockade by monoclonal antibodies, synthetic peptides, or snake venoms are encouraging, several problems require consideration (reviewed by Coller and associates,100 Ruggeri,101102 and Coller104). In addition to the potential for developing bleeding complications, immunogenicity is of concern, particularly with regard to the use of whole murine monoclonal antibodies, as is achieving effective doses of peptides or snake venoms due to their short halflives in the circulation. Furthermore, selectivity is an important consideration as integrin receptors are highly homologous and widely distributed among cells of different tissues. These receptors recognize RGD-containing proteins and peptides and participate in many processes of biological relevance, including immune reactions and other defense mechanisms. Thus the development of specific, platelet-selective agents for use in antithrombotic therapy remains an important goal.

PEERSCHKE Platelet Glycoproteins

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surface membrane glycoproteins involved in adhesive interactions. Not only is the molecular biology of congenital bleeding disorders becoming clearer, but also better assays for carrier detection and antenatal diagnosis are on the horizon. In addition, new antiplatelet therapies, based on specific platelet membrane glycoprotein receptor blockade, using either monoclonal antibodies or synthetic peptides or snake venoms, may have significant therapeutic advantages over current approaches. Furthermore, the availability of monoclonal antibodies directed against activation-dependent epitopes has implications for the diagnosis of prethrombotic and thrombotic states. The potential specificity of monoclonal antibodies also suggests future applications, including the targeting of thrombolytic and antithrombotic agents to areas of vessel occlusion by chemically cross-linking relevant antibodies with appropriate enzymes or inhibitors.

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