Platelet Membrane Receptor Research Articles

Functional property of von Willebrand factor under flowing blood.

von Willebrand factor (vWF) is produced in megakaryocytes and endothelial cells, is stored in the alpha-granule of platelets and in the Weibel-Palade body of endothelial cells, and is present in plasma and vascular subendothelium. This huge protein with a unique multimeric structure plays a pivotal role in both hemostasis and pathological intravascular thrombosis, in which vWF contributes to both platelet adhesion/aggregation and blood coagulation through its multiple adhesive functions for the platelet membrane receptors, glycoprotein Ib-IX-V complex, integrin alphaIIbbeta3, heparin, various types of collagen, and coagulation factor VIII. Among various functions, the most characteristic feature of vWF is its determinant role on platelet thrombus formation under high-shear-rate conditions. Indeed, at in vivo rheological situations where platelets are flowing with high speed in the bloodstream, the only reaction that can initiate mural thrombogenesis is the interaction of vWF with platelet glycoprotein Ibalpha. The recent x-ray analysis of the crystal structure of various functional domains and functional studies of this protein under experimental flow conditions have rapidly advanced and revised our knowledge of the structure-function relationships of vWF, a key protein for hemostasis and arterial thrombosis.

Clumping Factor A Mediates Binding of Staphylococcus aureus to Human Platelets

The direct binding of bacteria to platelets may be an important virulence mechanism in the pathogenesis of infective endocarditis. We have previously described Staphylococcus aureus strain PS12, a Tn551-derived mutant of strain ISP479, with reduced ability to bind human platelets in vitro. When tested in an animal model of endocarditis, the PS12 strain was less virulent than its parental strain, as measured by bacterial densities in endocardial vegetations and incidence of systemic embolization. We have now characterized the gene disrupted in PS12 and its function in platelet binding. DNA sequencing, Southern blotting, and PCR analysis indicate that PS12 contained two Tn551 insertions within the clumping factor A (ClfA) locus (clfA). The first copy was upstream from the clfA start codon and appeared to have no effect on ClfA production. The second insertion was within the region encoding the serine aspartate repeat of ClfA and resulted in the production of a truncated ClfA protein that was secreted from the cell. A purified, recombinant form of the ClfA A region, encompassing amino acids 40 through 559, significantly reduced the binding of ISP479C to human platelets by 44% (P = 0.0001). Immunoprecipitation of recombinant ClfA that had been incubated with solubilized platelet membranes coprecipitated a 118-kDa platelet membrane protein. This protein does not appear to be glycoprotein IIb. These results indicate that platelet binding by S. aureus is mediated in part by the direct binding of ClfA to a novel 118-kDa platelet membrane receptor.

Binding affinity of a newly synthesized 5-HT2 antagonist, AT-1015 (N-[2-[4-(5H-Dibenzo[a,d]cyclohepten-5-ylidene)-piperidino]ethyl]-1-formyl-4-piperidinecarboxamide monohydrochloride monohydrate), in the rabbit platelet membrane.

The object of this study was to investigate the binding affinity of a newly synthesized 5-HT2 antagonist, (N-[2-[4-(5H-dibenzo[a,d]cyclohepten-5-ylidene)-piperidino]ethyl]-1-formyl-4-piperidinecarboxamide monohydrochloride monohydrate) (AT-1015), in the rabbit platelet membrane using [3H]-ketanserin by radioligand binding assay method and to compare the results with other selective 5-HT2 antagonists. The results showed that AT-1015 displayed high affinity to 5-HT2 receptors in rabbit platelet membranes. The pKi value of AT-1015 was 7.40, which is slightly lower than that of ketanserin, but higher than that of cyproheptadine. On the other hand, the displacement potency of AT-1015 for 5-HT2 receptors in rabbit platelets was similar to those of sarpogrelate and ritanserin. This is the first report of the high affinity of AT-1015 in rabbit platelets.

Molecular Requirements in the Recognition of Low-Density Lipoproteins (LDL) by Specific Platelet Membrane Receptors

We have demonstrated that platelet low-density lipoprotein (LDL) receptors differ from classic LDL receptors of nucleated cells. Although positively charged Arg and Lys residues of apoprotein B-100 are known to play a key role in LDL recognition by classic LDL receptors, there are no conclusive data on platelet LDL receptors. This study investigated the molecular requirements of LDL particle recognition by platelet LDL receptors. The involvement of lipid and protein fractions was determined by displacement studies of the binding of 125I-LDL to platelets and fibroblasts (used as a classical LDL receptor model). The role of the protein moiety was evaluated by chemically modifying positively charged apoB residues (Lys, Arg, and Tyr) via copper-induced oxidation, cyclohexanedione, and tetranitromethane, respectively. The involvement of the lipid fraction was determined by ligand binding assays using 125I-LDL particles that had previously been delipidated and subjected to apoB solubilization. The degree of particle modification was analyzed by agarose/acrylamide gel electrophoresis and anion exchange chromatography. Modifying the amino acid residues increased particle electronegativity in the following order of potency: CHD-LDL>TNM-LDL>ox-LDL>native LDL. The results obtained by displacement studies in fibroblasts suggested that the gain in the LDL negative charge was the most important factor in the loss of receptor affinity. The chemical models of protein modification used in our study greatly affected LDL binding to the classical fibroblast receptor. In contrast, there was very slight difference in the displacement capacity on platelet 125I-LDL binding, which suggests that the protein fraction does not play a major role in the interaction of LDL with its platelet receptor. On the other hand, whereas modifying the lipid moiety did not alter the ability of solubilized 125I-apoB to interact with the classical fibroblast LDL receptor, platelet LDL receptors were unable to recognize these particles. In conclusion, our results confirm that the protein fraction plays a key role in the fibroblast LDL-receptor recognition process, whereas the lipid fraction appears to have a more relevant role in platelet LDL-receptor recognition.

Arteriosclerosis and the promise of GPIIb/IIIa inhibitors in stroke.

Ischemic mechanisms in pa2000Mar;58(1)1-10 studied for more than 150 years. Antiplatelet agents did show benefit in secondary prevention. Aspirin is the most common antiaggregant in clinical use today. However, the benefit produced by the "best" antiplatelet regimen in stroke prevention is lower than 40%. The adherence of circulating platelets to the subendothelium is mediated by glycoprotein (GP) residing on the cell's surface. GPIIb/IIIa is the most important platelet membrane receptor that mediates the process of platelet aggregation, and thrombus formation. Thus, new drugs that block the GPIIb/IIIa receptor have recently emerged. Clinical trials using these agents have shown effectiveness in acute coronary syndromes. However, the absence of studies in cerebrovascular disease and the potential hemorrhagic complications questioned their use in stroke prevention. We review the clinical trials using the new GPIIb/IIIa agents in myocardial ischemia, and consider the potential implications for cerebrovascular disease.

DDAVP is not a panacea for children with bleeding disorders.

DDAVP is not a panacea for children with bleeding disorders.

Platelet Peripheral-Type Benzodiazepine in Pregnancy and Lactation

The aim of the present study was to investigate the impact of hormonal changes during pregnancy and lactation on the expression of peripheral-type benzodiazepine receptors in platelet membranes. Platelet peripheral benzodiazepine receptor binding characteristics, Hamilton anxiety and depression rating Scores, and progesterone and prolactin (PRL) levels were evaluated during pregnancy and lactation in 17 pregnant women [first (n = 9) and third (n = 8) trimesters], 10 lactating women, and 8 nonpregnant women. A significant decrease (38–41%) in peripheral benzodiazepine receptor density was observed in women during the third trimester of pregnancy when compared to nonpregnant controls and women in their first trimester of pregnancy. The decrease is peripheral benzodiazepine receptors was parallel to the peak in progesterone and PRL secretion. The reduction in peripheral benzodiazepine receptor expression is hormone-dependent and may play a regulatory role geared to prevent pregnancy-related overactivity of the hypothalamic–pituitary-ovarian, hypothalamic–pituitary-adrenal, and hypothalamic-PRL axes.

Von Willebrand Factor Receptor GPIbα is Expressed by Human Factor XIIIa-Positive Dermal Dendrocytes and is Upregulated by Mast Cell Degranulation

GPIb alpha, a glycoprotein component of the GPIb-IX-V complex, serves as a platelet membrane receptor that mediates adhesion to von Willebrand factor normally present in the vascular subendothelium. Recent data have demonstrated that GPIb alpha is not restricted to platelets, but is also expressed by endothelium in vitro. In this study, we describe the expression and distribution of GPIb alpha in normal adult and neonatal human skin. GPIb alpha is present, as detected by immunohistochemistry, on endothelial cells and on highly dendritic cells localized within the perivascular space, dermal-epidermal junction, and reticular dermis. By dual-labeling immunofluorescence and confocal microscopy, GPIb alpha-positive cells within the dermal interstitium are demonstrated to represent factor XIIIa-positive dermal dendrocytes. In organ cultures of neonatal human foreskin, mast cell degranulation induced by either substance P or compound 48/80 resulted in transiently increased GPIb alpha expression by dermal dendrocytes. Because the GPIb-IX-V complex plays a part in regulating hemostasis and may be important for cellular interactions with extracellular matrix molecules, these data provide additional insight into the potential function of FXIIIa-positive dermal dendrocytes in skin remodeling and repair.

Nonfractionated Heparin Fails to Inhibit Arterial Thrombosis in a Human Ex Vivo Thrombosis Model

The effect of nonfractionated heparin on the formation and composition of arterial thrombus is unclear. The purpose of this study in a human ex vivo model was to analyze fibrinoplatelet thrombi and test the inhibitory effect of nonfractionated heparin on arterial thrombus formation. Experiments were carried out in Sakariassen perfusion chambers. Strips coated with either tissue factor (TF) or collagen were exposed to human blood collected from healthy volunteers at an arterial shear stress rate of 2600 s-1 for 1 to 4 min. Platelet deposition was determined using immunoenzymatic techniques to quantify P-selectine, a platelet membrane receptor, in thrombi. Fibrin deposition was determined by quantifying fibrin degradation products released after application of plasmin (D-dimers). Heparin was injected into the blood flow through a blender port system located between the venous puncture site and perfusion chamber. The results of the study showed that in a human ex vivo model, formation of arterial thrombus on two thrombogenic surfaces (tissue factor and collagen) is not inhibited by nonfractionated heparin.

Platelet-derived microparticles on synthetic surfaces observed by atomic force microscopy and fluorescence microscopy.

Platelet activation on a thrombogenic surface includes the release of membrane-derived microparticles that provide catalytic sites for blood coagulation factors. Here, we describe a quantitative investigation on the production and dimensions of platelet-derived microparticles observed on glass and polyethylene under aqueous conditions, using atomic force microscopy (AFM) and complementary fluorescence microscopy. The results show that contact-activated platelet microparticles are not evenly distributed over a thrombogenic surface, but in clusters in close proximity to adherent platelets. The microparticles are localized near the platelet periphery, and in some cases appear to emanate from platelet pseudopodia, suggesting that formation may result from vesiculation of the pseudopodia. The microparticles measured 125 +/- 21 nm (n = 73) in the x-y dimensions and 5.2 +/- 3.6 nm in height. The results compared closely with 125 +/- 22 nm width and 4.1 +/- 1.6 nm height obtained for control preparations of thrombin activated microparticles, that were filtered and deposited on glass. Large differences between the measured widths and heights of adsorbed microparticles suggest that platelet microparticles may undergo spreading after attachment to a surface. The adsorbed microparticles expressed platelet membrane receptor GPIIb/IIIa, and many expressed the platelet activation marker P-selectin as determined by fluorescence microscopy. The high number distribution of procoagulant microparticles per unit area of surface compared with platelets suggests that platelet-derived microparticles provide a mechanistic route for amplifying thrombus formation on a thrombogenic surface.

Vitamin B6 down-regulates the expression of human GPIIb gene.

Glycoprotein IIb (GPIIb) is the alpha-subunit of the platelet membrane receptor GPIIb/IIIa, which plays a major role in platelet aggregation. Vitamin B6 has been reported to be an antiaggregative agent, although its mechanism is not well known. To understand the molecular mechanism of vitamin B6 on antiaggregation, we analyzed the effects of various forms of vitamin B6 on the expression of human GPIIb promoter using chloramphenicol acetyl transferase (CAT) as the reporter gene. The GPIIb promoter region was amplified by polymerase chain reaction (PCR), cloned into pBLCAT3 to drive the CAT reporter gene and transfected into human erythroleukemia (HEL) cells. Transient expression of the GPIIb promoter was determined after transfected cells were treated with 1 microM pyridoxine (PN), pyridoxal (PL), pyridoxal-5-phosphate (PLP), or 4-deoxypyridoxine (4-dex) for 48 h. Our results show that the GPIIb promoter activity was down-regulated to 54, 35 and 63% in the presence of PN, PL and PLP, respectively, as compared to an untreated control whose promoter activity was 100%. However, no adverse effect on GPIIb promoter was detected by 4-dex, which is an antagonist of vitamin B6. The down-regulation effect of vitamin B6 on GPIIb promoter activity may lead to a reduction of GPIIb protein expression and thus be detrimental to platelet aggregation.

Influence of Low- and High-Dose Aspirin Treatment on Thrombin Generation in Whole Blood

The effects of two doses of aspirin (75 and 500 mg/day during 1 week) on thrombin generation was investigated in healthy volunteers. Thrombin generation in whole blood was monitored by repeated measurements of prothrombin fragment 1+2 (F1+2) in plasma prepared from untreated whole blood left to clot at 37°C. Experiments with a platelet inhibiting agent (iloprost, a prostacyclinanalogue) and platelet-activating compounds (collagen and a thromboxane analogue), indicated that the formation of thrombin in this system is partly dependent on platelet function. High dose aspirin (500 mg daily) attenuated thrombin generation, whereas low-dose treatment (75 mg daily) failed to attenuate thrombin formation significantly. Collagen-induced platelet aggregation in whole blood, used to monitor antiplatelet effects of aspirin, showed profound inhibition of platelet aggregation already at 75 mg of aspirin; 500 mg did not inhibit platelet aggregation further. Our results show that aspirin suppresses thrombin formation in whole blood in a dose-dependent fashion and that the “antithrombin” effects of aspirin require higher doses than the antiaggregating effects. The mechanism(s) behind the “antithrombin” effects of aspirin is at present unclear but may involve thromboxane-independent mechanisms, such as acetylation of platelet membrane receptors or coagulation factors.

Analysis of the Three Dimensional Relationships Among Components of Human Vascular Subendothelium by Stereo-Tilt Immunoelectron Microscopy

Stereo-tilt immunoelectron microscopy offers a method for analyzing the inter-relationships among components of the subendothelium at the 3 dimensional level. It has been demonstrated that von Willebrand factor (vWF) is important in the process of platelet adhesion following endothelial injury by serving as a bridge between constituents of the vascular subendothelium and platelet membrane receptors. We had previously shown that type VI collagen microfibrils serve as a binding site for vWF in human vascular subendothelium, and immunolabeling studies of vascular tissues by routine transmission electron microscopy showed apparent co-localization of the 2 proteins.We investigated the relationships among vWF, fibrillin and type VI collagen in human vascular subendothelium with double labeling immunogold localization techniques in conjunction with stereo-tilt electron microscopy to examine the 3 dimensional ultrastructure of vWF-microfibril complexes.Our studies confirmed that vWF co-localizes with the type VI collagen microfibrils in subendothelium and not with fibrillin as suggested by others.The vWF is present in subendothelium in the form of electron-dense aggregates having diameters varying between 65–80 nm that decorate the type VI collagen microfibrils and having structural similarities to intracellular Weibel- PaIade bodies. The 3 dimensional representations show that the labeling of the 2 molecular species are not necessarily directly adjacent to one another and can be some distance from each other. These results confirm our previous observations on the association between type VI collagen and vWF. Importantly, they also show that colocalization studies where immunolabeling is performed in situ and observed by electron microscopy must be evaluated 3 dimensionally before one can determine conclusively that the 2 components are associated with one another. (The J Histotechnol 21:205–212, 1998)

Oral inhibitors of platelet membrane receptor glycoprotein IIb/IIIa in clinical cardiology: Issues and opportunities

The feedback amplification loop in the progress of medicine, from basic findings and clinical observations to hypothesis generation and new research, is presently particularly dynamic in acute coronary syndromes. As the role of thrombosis formation in etiopathology of these syndromes has emerged, antithrombotic therapy has become standard practice. Numerous trials with antiplatelet drugs and meta-analyses of these trials have established the efficacy of aspirin, launching new research in platelet physiology, understanding of pathophysiology, and therapy. Recognition of the role of glycoprotein IIb/IIIa receptor in health and disease and of its dynamic and ligands lead to production of a monoclonal antibody against the receptor, which has become the first anti-integrin therapy approved for clinical use. Synthetic compounds were subsequently produced containing the specific arginine-glycine-aspartic acid (RGD) sequence for fibrinogen binding to the receptor. Inhibitors are now available for oral use, allowing prolonged blockade of the receptor and opportunities for new research and hypothesis generation as well as better patient care. This supplement to the American Heart Journal summarizes a workshop whose goal was to generate a discussion among fundamentalists and clinicians on methods for monitoring platelet function with these drugs and investigation of consequences of chronic receptor occupancy. (Am Heart J 1998;135:S107-S112.)

Physical Proximity and Functional Interplay of the Glycoprotein Ib-IX-V Complex and the Fc Receptor FcγRIIA on the Platelet Plasma Membrane

Although the glycoprotein (GP) Ib-IX-V complex and FcgammaRIIA are distinct platelet membrane receptors, previous studies have suggested that these structures may be co-localized. To determine more directly the proximity of GP Ib-IX-V and FcgammaRIIA, we assessed the effects of anti-GP Ibalpha monoclonal antibodies on FcgammaRIIA-mediated platelet aggregation and on the direct binding of polymeric IgG to human platelets. In addition, we directly examined the proximity of FcgammaRII and GP Ib-IX-V using flow cytometric fluorescence energy transfer and immunoprecipitation studies. Preincubation of platelets with either of two monoclonal antibodies (AN51 or SZ2) directed against GP Ibalpha completely blocked platelet aggregation by polymeric IgG. Similarly, these antibodies totally inhibited platelet aggregation by two strains of viridans group streptococci known to induce aggregation via FcgammaRIIA. In addition, AN51 and SZ2 significantly reduced the binding of polymeric IgG to washed fixed platelets. When assessed by flow cytometry, significant levels of bidirectional energy transfer were detected between FcgammaRIIA and GP Ibalpha, indicating a physical proximity of less than 10 nm between these receptors. This energy transfer was not due to high receptor density, because no homoassociative energy transfer was seen. Moreover, immunoprecipitation of FcgammaRIIA from platelet lysates also co-precipitated GP Ibalpha. These results indicate that GP Ibalpha and FcgammaRIIA are co-localized on the platelet membrane and that this association is not random.

Increased Potency and Decreased Elimination of Lamifiban, a GPIIb-IIIa Antagonist, in Patients with Severe Renal Dysfunction

Activation of the platelet membrane receptor glycoprotein (GP) IIb-IIIa is essential for thrombus formation. The novel nonpeptide GPIIb-IIIa antagonist, lamifiban, represents a promising approach for antiplatelet therapy in patients with cardiovascular disease. Since renal impairment frequently occurs in these patients, we designed a phase I study to assess the tolerability, pharmacodynamics and pharmacokinetics of lamifiban in patients with renal impairment. Four healthy volunteers (Group 1) with creatinine clearance (CLCR) >75 ml/min, eight patients (Group 2) with mild to moderately impaired renal function (CLCR 30-74 ml/min) and eight patients (Group 3) with severe renal impairment (CLCR 10-29 ml/min) were studied. They received stepwise increased doses of lamifiban intravenously (i.v.). There was a linear relationship between the systemic clearance of the drug and renal function (R2 = 0.86). The mean plasma concentration required for half-maximal inhibition of thrombin-receptor agonist peptide (TRAP) induced platelet aggregation (EC50) ex vivo was 21, 28 and 11 ng/ml in Groups 1, 2 and 3. The patients in Group 3 were sensitized to the antiplatelet effect allowing an 18-fold dosage reduction without compromising the pharmacodynamics. In conclusion, the decreased clearance of lamifiban may act in concert with increased potency of the drug in patients with severe renal impairment, and the drug dosage should be reduced accordingly.

Polymorphisms of HPA-1 Through 6 on Platelet Membrane Glycoprotein Receptors Are Not a Genetic Risk Factor for Myocardial Infarction in the Japanese Population

We examined HPA genotypes derived from polymorphisms of platelet membrane receptors in 88 Japanese patients with early-onset myocardial infarction and in 100 control subjects. The results indicated that HPA genotypes 1 through 6 are not associated with early-onset myocardial infarction.

Intracellular Inositol(1,4,5)-Trisphosphate Receptor Levels Are Preserved in Alzheimer’s Disease Platelets

An increasing number of signal transduction disturbances have been reported in Alzheimer’s disease. These changes are not restricted to histopathologically changed brain areas but are seen also in peripheral tissues. One of the most severe disturbances is a loss of calcium-mobilizing intracellular inositol(1,4,5)-trisphosphate receptors in Alzheimer cerebellar and cortical tissues. In the present study, the binding of [ 3H]inositol(1,4,5)trisphosphate ([ 3H]Ins(1,4,5)P 3) to the calcium-mobilizing inositol(1,4,5)trisphosphate receptors in platelet membranes from eight Alzheimer’s disease patients and eight control subjects were investigated to determine its possible role as a biological marker in Alzheimer’s disease. It was found that there were no significant difference in [ 3H]Ins(1,4,5)P 3 binding with respect to the number of sites measured at different protein concentrations or to the sensitivity of the binding to inhibition by nonradioactive Ins(1,4,5)P 3 between Alzheimer disease platelets and controls. It is concluded that inositol(1,4,5)trisphosphate receptor levels are preserved in platelets from patients with Alzheimer’s disease.

BINDING OF THE VON WILLEBRAND FACTOR A1 DOMAIN TO HISTONE

Activation of the von Willebrand Factor (vWF) A1 domain is a critical factor in regulating the interaction of vWF with its platelet membrane receptor, the glycoprotein (GP) Ib-IX-V complex. This activation controls vWF-dependent platelet adhesion at high shear. The vWF-GP Ib-IX-V interaction is induced in vivo by exposure of platelet-rich plasma to high shear force, or by association of vWF with one or more unidentified components of the subendothelial matrix. In vitro, soluble vWF is activated to bind to platelets by nonphysiological modulators, such as the bacterial glycopeptide, ristocetin, or the snake venom protein, botrocetin, or by removal of negatively-charged sialic acid residues. Analysis of vWF modulators and the very marked charge asymmetry of amino acid sequences within the A1 domain has led to an electrostatic model for vWF modulation. Endothelial membrane/matrix and detergent-soluble fractions of human placenta were screened for the ability to bind vWF by electrophoresis of extracts on SDS-polyacrylamide gels, electrotransferring to nitrocellulose and probing with fluid-phase 125I-labeled vWF or a 39/34-kDa vWF fragment (Leu-480-Gly-718) that encompasses the A1 domain. In the course of these studies, it was found that both vWF and the 39/34-kDa vWF fragment bound strongly to histone. Purified soluble histone also bound vWF since, like ristocetin, it induced vWF flocculation. Histone binding to vWF did not activate or inhibit vWF binding to platelets. While the vWF-histone interaction has no conceivable physiological role, it suggests that binding to the A1 domain of vWF alone is insufficient to modulate vWF adhesive activity. This implies that specific interactions of the vWF A1 domain with either ristocetin or botrocetin are required for GP Ib-IX-V recognition to occur. © 1997 Elsevier Science Ltd

Alternative expression of platelet glycoprotein Ib(beta) mRNA from an adjacent 5' gene with an imperfect polyadenylation signal sequence.

Glycoprotein (GP) Ib is a major component of the platelet membrane receptor for von Willebrand factor, designated the GP Ib-IX-V complex. GP Ib is composed of two subunits (GP Ib(alpha) and GP Ib(beta)) each synthesized from separate genes. The 206 amino acid precursor of GP Ib(beta) is synthesized from a 1.0-kb mRNA expressed by megakaryocytes and was originally characterized from cDNA clones of human erythroleukemic (HEL) cell mRNA, a cell line exhibiting megakaryocytic-like properties. The cell line CHRF-288-11 also exhibits megakaryocytic-like properties, but synthesizes two related GP Ib(beta) mRNA species of 3.5 and 1.0 kb. We performed cDNA cloning experiments to identify the origin of the 3.5-kb transcript and determine its relationship to the 1.0-kb GP Ib(beta) mRNA found in megakaryocytes, platelets, and HEL cells. Our cloning experiments demonstrate that the longer transcript results from a nonconsensus polyadenylation recognition sequence, 5'AACAAT3', within a separate gene located upstream to the platelet GP Ib(beta) gene. In the absence of normal polyadenylation the more 5' gene uses the polyadenylation site within its 3' neighbor, the platelet GP Ib(beta) gene. This newly identified 5' gene contains an open reading frame encoding 369 amino acids with a high degree of sequence similarity to an expanding family of GTP-binding proteins.