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Clustering and occupancy of platelet integrin αIIbβ3(GPIIb-IIIa) generate biologically important signals: conversely, intracellular signals increase the integrins’ affinity, leading to integrin activation; both forms of integrin signaling play important roles in hemostasis and thrombosis. Indirect evidence implicates interactions between integrin α and β transmembrane domains (TMDs) and cytoplasmic domains in integrin signaling; however, efforts to directly identify these associations have met with varying and controversial results. In this study, we develop mini-integrin affinity capture and use it in combination with nuclear magnetic resonance spectroscopy to show preferential heterodimeric association of integrin αIIbβ3TMD tails via specific TMD interactions in mammalian cell membranes in lipid bicelles. Furthermore, charge reversal mutations at αIIb(R995)β3(D723) confirm a proposed salt bridge and show that it stabilizes the TMD-tail association; talin binding to the β3tail, which activates the integrin, disrupts this association. These studies establish the preferential heterodimeric interactions of integrin αIIbβ3TMD tails in mammalian cell membranes and document their role in integrin signaling.

Agonists induce inside-out αIIbβ3signaling resulting in fibrinogen binding and platelet aggregation. These in turn trigger outside-in signaling resulting in further platelet stimulation. Because the Syk tyrosine kinase is activated during both phases of integrin signaling, we evaluated its role in αIIbβ3function in murine platelets rendered null for Syk by gene targeting and in human platelets incubated with piceatannol, a tyrosine kinase inhibitor reportedly selective for Syk. Both Syk null murine platelets and piceatannol-treated human platelets exhibited a partial, but statistically significant defect in activation of αIIbβ3by adenine diphosphate (ADP) ± epinephrine as assessed by fibrinogen binding. Syk null platelets adhered normally to immobilized fibrinogen, and mice with these platelets exhibited normal tail bleeding times. In contrast, piceatannol treatment of human platelets completely inhibited platelet adhesion to immobilized fibrinogen. The discrepancy in extent of integrin dysfunction between murine and human platelet models may be due to lack of specificity of piceatannol, because this compound inhibited the activity of Src and FAK as well as Syk and also reduced tyrosine phosphorylation of multiple platelet proteins. These results provide genetic evidence that Syk plays a role in αIIbβ3signaling in platelets and pharmacological evidence that, although piceatannol also inhibits αIIbβ3signaling, it does so by inhibtion of multiple protein tyrosine kinases.

Agonists induce inside-out ?IIbß3signaling resulting in fibrinogen binding and platelet aggregation. These in turn trigger outside-in signaling resulting in further platelet stimulation. Because the Syk tyrosine kinase is activated during both phases of integrin signaling, we evaluated its role in ?IIbß3 function in murine platelets rendered null for Syk by gene targeting and in human platelets incubated with piceatannol, a tyrosine kinase inhibitor reportedly selective for Syk. Both Syk null murine platelets and piceatannol-treated human platelets exhibited a partial, but statistically significant defect in activation of ?IIbß3 by adenine diphosphate (ADP) ± epinephrine as assessed by fibrinogen binding. Syk null platelets adhered normally to immobilized fibrinogen, and mice with these platelets exhibited normal tail bleeding times. In contrast, piceatannol treatment of human platelets completely inhibited platelet adhesion to immobilized fibrinogen. The discrepancy in extent of integrin dysfunction between murine and human platelet models may be due to lack of specificity of piceatannol, because this compound inhibited the activity of Src and FAK as well as Syk and also reduced tyrosine phosphorylation of multiple platelet proteins. These results provide genetic evidence that Syk plays a role in ?IIbß3 signaling in platelets and pharmacological evidence that, although piceatannol also inhibits ?IIbß3 signaling, it does so by inhibtion of multiple protein tyrosine kinases.

An unbiased genetic approach was used to identify a specific amino acid residue in the αIIbsubunit important for the ligand binding function of the integrin αIIbβ. Chemically mutagenized cells were selected by flow cytometry based on their inability to bind the ligand mimetic antibody PAC1 and a cell line containing a single amino acid substitution in αIIbat position 224 (D→V) was identified. Although well expressed on the surface of transfected cells, αIIbD224Vβ3as well as αIIbD224Aβ3did not bind αIIbβ3-specific ligands or a RGD peptide, a ligand shared in common with αvβ3. Insertion of exon 5 of αIIb, residues G193-W235, into the backbone of the αvsubunit did not enable the chimeric receptor to bind αIIbβ3-specific ligands. However, the chimeric receptor was still capable of binding to a RGD affinity matrix. αIIbD224 is not well conserved among other integrin α subunits and is located in a region of significant variability. In addition, amino acid D224 lies within a predicted loop of the recently proposed β-propeller model for integrin α subunits and is adjacent to a loop containing amino acid residues previously implicated in receptor function. These data support a role for this region in ligand binding function of the αIIbβ3receptor.

An unbiased genetic approach was used to identify a specific amino acid residue in the ?IIb subunit important for the ligand binding function of the integrin ?IIbß. Chemically mutagenized cells were selected by flow cytometry based on their inability to bind the ligand mimetic antibody PAC1 and a cell line containing a single amino acid substitution in ?IIb at position 224 (D?V) was identified. Although well expressed on the surface of transfected cells, ?IIbD224Vß3 as well as ?IIbD224Aß3 did not bind ?IIbß3-specific ligands or a RGD peptide, a ligand shared in common with ?vß3. Insertion of exon 5 of ?IIb, residues G193-W235, into the backbone of the ?v subunit did not enable the chimeric receptor to bind ?IIbß3-specific ligands. However, the chimeric receptor was still capable of binding to a RGD affinity matrix. ?IIbD224 is not well conserved among other integrin ? subunits and is located in a region of significant variability. In addition, amino acid D224 lies within a predicted loop of the recently proposed ß-propeller model for integrin ? subunits and is adjacent to a loop containing amino acid residues previously implicated in receptor function. These data support a role for this region in ligand binding function of the ?IIbß3 receptor.

Fibrinogen, fibronectin, von Willebrand factor, and thrombospondin are four large glycoproteins that bind to thrombin-stimulated platelets and influence cellular adhesive functions. The effects of five monoclonal antibodies that react with platelet membrane glycoproteins (GP) IIb and/or IlIa on the binding of these four molecules to stimulated platelets were assessed. Tab and PMI-1, antibodies recognizing GPIIb, had no effect, whereas 10E5 and 2G12, antibodies that immunoprecipitate both GPIIb and IIIa in the presence of calcium, inhibited binding of all four ligands by >85%. T10, an antibody specific for the GPIIb—IIIa complex, produced partial inhibition (60% to 80%) of the binding of each ligand. Inhibitory antibodies were effective in the same dose range for all four proteins and also inhibited binding of fibrinogen, fibronectin, and von Willebrand factor to receptors fixed in an induced state (thrombin-stimulated platelets fixed with paraformaldehyde). Thrombospondin did not bind to these fixed cell preparations. The results suggest that these four adhesive proteins have a related mechanism of binding to thrombin-stimulated platelets. This related mechanism may entail the sharing of some, but not necessarily all, binding sites for the four ligands or a proximal relationship between these binding sites.

Cellular fibronectin (Fn) bearing an alternatively spliced extra type III structural repeat (ED1) is normally present at low concentrations in blood plasma. The source of this material remains uncertain. In this study, primary cultures of human umbilical vein endothelial cells (HUVEC) labeled with 35S-methionine were observed to synthesize Fn monomers both with and without this segment. Monomers isolated from cell lysates with antibodies to the ED1 sequence comigrated in nonreduced sodium dodecyl sulfate polyacrylamide gel electrophoresis with the slower (designated M1). but not the faster (designated M2), of two major monomeric populations that were recognized by antibodies raised to plasma-derived Fn. The differences between M1 and M2 were not due to glycosylation, since they were also observed between species of Fn monomer purified from cells grown in the presence of tunicamycin. M1 and M2 were both observed to incorporate with a similar rate into dimeric Fn, indicating that Fn monomers with and without the ED1 site can dimerize with similar efficiency. Analysis of reduced samples of Fn isolated from cells with anti-ED1 antibodies indicated the presence of both M1-M1 and M1-M2 dimers. In addition to being incorporated into extracellular matrix, ED1 + Fn was observed to be secreted in soluble form into the medium, potentially reflecting intravascular release of this protein by endothelial cells in vivo.

The multimeric structure of platelet factor VIII/von Willebrand factor (FVIII/vWF) in cell extracts and in collagen and thrombin releasates has been analyzed by SDS polyacrylamide gel electrophoresis followed by detection with 125I-anti-FVIII/vWF. Platelets contained larger multimers than those normally present in plasma. When secreted FVIII/vWF was analyzed, all platelet forms were released from collagen-stimulated platelets. In contrast, in thrombin releasates the larger multimers were lost in a manner dependent on divalent cations, time, and thrombin dose. This loss could not be accounted for by modification of FVIII/vWF by thrombin or platelet enzymes since no effect of thrombin on the multimeric structure of FVIII/vWF in the absence of platelets or in the presence of platelet lysates was observed. Large multimers of 125I-labeled purified FVIII/vWF underwent divalent cation-dependent association with platelets in the presence of thrombin, indicating that the loss of FVIII/vWF from thrombin releasates was due to reassociation with the platelet. These studies show a structural difference between platelet and plasma FVIII/vWF that suggests a specific role for platelet FVIII/vWF in hemostasis.

We have investigated the localization of thrombospondin (TSP), fibrinogen, fibronectin, and von Willebrand factor in human platelets by transmission electron microscopy of antibody-stained ultrathin frozen sections. In negatively stained thin sections, alpha granules were identified on the basis of their smooth, roughly spherical shape, size, single limiting electron-lucent 100 Å membrane, and frequent presence of electron-dense nucleoid. In contrast, mitochondria exhibited characteristic double membranes and cristae. Sections were separately stained with affinity-purified polyclonal antibodies to these proteins as well as with three monoclonal anti-TSP antibodies. Antibody specificity was documented in radioimmunoassays, by immunofluorescent cross-blocking, and by staining of bands of appropriate mobility in Western blots of whole platelets. Bound antibody was visualized using a 5-nm colloidal gold-avidin conjugate. In resting cells, staining of virtually all alpha granules was observed for all four proteins. In contrast, consistent staining was absent from other organelles, including plasma membranes, mitochondria, and vacuolar structures that may represent the open canalicular system.

To analyze molecular mechanisms of platelet aggregation, we have studied the aggregation of Chinese hamster ovary (CHO) cells expressing between 1 and 4 × 105recombinant human glycoprotein (GP) llb-lll, molecules per cell (A5 cells). These cells aggregated as measured by the disappearance of single cells during rotary agitation. Aggregation was dependent on the presence of extracellular fibrinogen ( = 500 nmol/L) and divalent cations, and required prior activation of the GPIIb-llla. A synthetic peptide (GRGDSP) and monoclonal anti-GPIIb-lllaantibody (2G12) that block platelet aggregation also blocked aggregation of these cells. Parent CHO cells or those expressing recombinant GPIIb-lllacontaining a point mutation that causes variant thrombasthenia both failed to aggregate when stimulated in the presence of fibrinogen. These data show that GPIIb-lllais the only unique platelet surface component required for aggregation.

Plasma fibronectin binds in a specific and saturable manner to thrombin-stimulated platelets. 7-Thrombin stimulated 80% as much fibronectin binding to platelets as a-thrombin with conversion of ≤1% of platelet fibrinogen to fibrin. Afibrinogenemic and normal platelets bound similar quantities of fibronectin in the presence of calcium or magne-sium-ethylene glycol tetra-acetic acid (EGTA). These observations indicate that fibronectin can interact with platelets without involvement of fibrin or fibrinogen. Nevertheless, two different effects of fibrin(ogen) on fibronec-tin binding were observed. First, exogenous fibrinogen inhibited fibronectin binding to thrombin-stimulated platelets. This inhibition was unidirectional, as fibronectin did not inhibit fibrinogen binding to ADP or thrombin-stimu-lated cells. Second, formaldehyde-fixed cells with surface-associated fibrin bound significant quantities of fibronectin. This interaction required calcium and did not occur on fixed cells with or without surface-bound fibrinogen. A portion of the ligand bound to fixed cells with surface-associated fibrin was modified to form a derivative with a molecular weight identical to that of the fibronectin subunit crosslinked to the a-chain of fibrin. This high mol wt derivative was also observed to a variable extent with living cells in the presence of magnesium or calcium but not in the presence of magnesium-EGTA. Thus, fibronectin binds to platelets by at least two mechanisms: (1) a fibrin(ogen)-independent pathway that requires divalent ions and is inhibited by exogenous fibrinogen; and (2) a fibrin-depen-dent pathway with an absolute calcium requirement. With nonaggregated, thrombin-stimulated platelets, the former pathway appears to predominate. © 1985 by Grune & Stratton, Inc.

Normal primary platelet aggregation requires agonist-mediated activation of membrane GPIIb-IIIa, binding of fibrinogen to GPIIb-IIIa, and cellular events after ligand binding. PAC1 monoclonal antibody distinguishes between resting and activated states of GPIIb-IIIa, and other antibodies preferentially recognize GPMb (PMI-1) or Ilia (anti-LIBS1) after the binding of fibrinogen or fibrinogen-mimetic peptides, such as GRGOSP. Using these antibodies and platelet flow cytometry, we studied two distinct persistent platelet aggregation abnormalities. Platelets from a thrombasthenia variant, which contained near-normal amounts of GPIIb-IIIa, failed to aggregate or bind PAC1 in response to agonists. In addition, GRGDSP, which binds to normal GPIIb-IIIa without prior cell activation, failed to Increase the binding of PMI-1 or anti-LIBS1 to the thrombasthenia platelets, suggesting a primary defect In ligand binding. Chromatography of detergent-solubilized platelets on a KYGRGDS affinity column confirmed that the patient’s GPIIb-IIIa lacked the fibrinogen binding site. In another patient with myelofibrosis and defective aggregation, PAC1 failed to bind to adenosine diphosphate–stimulated platelets, but did bind when protein kinase C was directly activated with phorbol myristate acetate. Furthermore, the binding of PMI-1 and anti-LIBS1 increased in response to GRGDSP, confirming a defect in agonist-mediated fibrinogen receptor activation rather than in fibrinogen binding or events distal to binding. These studies indicate that this immunochemical approach is useful in classification of clinical abnormalities of platelet aggregation as defects in either (a) fibrinogen receptor activation, (b) fibrinogen binding, or (c) postoccupancy events.

Platelet membrane GPIIb is comprised of a disulfide-linked heavy chain (GPIIb(H)) and light chain (GPIIb(L)). We have examined the role of the two chains of GPIIb in the maintenance of the GPIIb-llla heterodimer and Arg-Gly-Asp (RGD) peptide-binding function. Lysates of surface radioio-dinated platelets were treated with 1 % 2-mercaptoethanol for 18 hours at 4°C. Reduction of the interchain disulfide in GPIIb was followed by immunoprecipitation with antipep-tide antibodies specific for GPIIb(H) or GPIIb(L). In addition to the GPIIb-llla complex, a polypeptide of 120 Kd was precipitated by anti-GPIIb(H) and a polypeptide of 23 Kd was precipitated by anti-GPIIb(L) from reduced platelet lysates. To determine whether GPIIb(H) or GPIIb(L) remained complexed with GPIIIa, reduced platelet lysates were immunoprecipitated with AP3, a monoclonal anti-GPIIIa antibody, resulting in the coimmunoprecipitation of GPIIb(H) but not GPIIb(L). Conversely, the monoclonal anti-GPIIb(H) antibody PMI-1 immunoprecipitated GPIIIa with GPIIb(H). Thus GPIIb(H) maintains its association with GPIIIa. Furthermore, the GPIIb(H)-llla complex retains its reactivity with AP2, a monoclonal antibody (MoAb) specific for the nondissociated GPIIb-llla complex. Affinity chromatography of reduced platelet lysates on immobilized KYGRGDS resulted in binding and specific elution of the GPIIb(H)-llla complex. These findings indicate that GPIIb(H) contains sufficient information for maintenance of a complex with GPIIIa and support of the binding of the heterodimer to RGD peptides.

The monoclonal antibody, OKM5, recognizes an 88-Kd monocyte membrane protein and also binds to the platelet membrane protein, GPIV (GPIIIb, CD36). In this study, we have found that the OKM5 target epitope is present at approximately 12,000 copies per platelet and that interaction with the antibody has both stimulatory and inhibitory effects on platelet function. In the absence of other stimuli, OKM5 induced platelet aggregation, secretion, and expression of fibrinogen receptors. These stimulatory responses required intact antibody as F(ab’)2fragments were not active but blocked the stimulatory activity of the intact antibody. In contrast, exposure of platelets to OKM5 followed by another strong stimulus such as thrombin resulted in a marked suppression of fibrinogen, fibronectin, and von Willebrand factor binding to the cells. This effect was not noted when a weak stimulus, adenosine diphosphate, was the second agonist. At OKM5 concentrations that interfered with fibrinogen binding to thrombin-stimulated platelets by 80% to 90%, platelet binding of exogenous thrombospondin, or surface expression of endogenous thrombospondin was not affected. The inhibitory effect of OKM5 on fibrinogen binding to thrombin-stimulated platelets was related to the formation of massive platelet aggregates in the samples. These results show that interaction of OKM5 with its target antigen on platelets can elicit diverse functional responses from the cells.

A biophysical approach was used to directly determine the avidity of the junction between two Chinese hamster ovary (CHO) cells bearing recombinant GpIIb-IIIa in the presence and absence of fibrinogen. Micromanipulation was used to induce conjugation of the cell pairs with or without activating the Gpllb-llla molecules with monoclonal antibody (MoAb) 62. Activation of Gpllb-llla caused an increase in the force required to separate the conjugates. The molecular bonding force between cells bearing activated Gpllb-llla and fibrinogen molecules was found to be 2.1 × 10−7dyne, which is 3.7 times higher than that between nonactivated Gpllb-IIIa and fibrinogen (5.7 × 10−8dyne). The results provide a quantitative assessment of the molecular bonding force between fibrinogen and the GpIIb-IIIa expressed on cell surface. The findings indicate that the activation of Gpllb-llla leads to an increase in the adhesive force in CHO cell aggregation by increasing the strength of the GpIIb-IIIa-fibrinogen bonds rather than the number of these bonds.

A platelet glycoprotein (GP) IlIa epitope library was constructed by insertion of randomly cleaved GPIIIa cDNA fragments in the prokaryotic expression vector λgt22 and screened with purified anti-PIA1antibodies for clones expressing a PIA1epitope. Five independent clones were isolated and characterized by nucleotide sequencing. The smallest anti-PIA1reactive clone obtained encoded the amino terminal 66 residues of mature GPIIIa. Substitution of leucine33(PIA1) with a proline33(PIA2) by in vitro mutagenesis resulted in the loss of anti-PIA1reactivity; however, this clone still reacted with anti-GPIIIa polyclonal antibodies. These data indicate that a PIA1alloantigenic epitope is located within a small, unglycosy-lated fragment of GPIIIa containing the polymorphism responsible for the PIAphenotype. Furthermore, these results prove that small recombinant mimics of a PIA1epitope may be synthesized and used for detection of these alloantibodies,1992 by The American Society of Hematology. 0006-4971/927903-0043$3.00/0

The distribution and transport in platelets of human coagulation Factor V was investigated by immunofluorescent and immunoelectron microscopy. In resting intact platelets, little surface staining was observed by immunofluorescence. In permeable resting cells, punctate staining similar to that reported for fibrinogen (Fbg), thrombospondin (TSP), fibronectin (Fn), von Willebrand factor (VWF), B-thromboglobulin (BTG), and platelet Factor 4 (PF4) was observed.1,2Double label immunofluorescent staining for Fbg and Factor V demonstrated colocalization, suggesting their presence in the same intracellular structure. Thrombin stimulation induced the appearance of larger (~0.5 µ)immunofluorescent masses of these proteins which exactly colocalized. Thus, at the light level, Factor V and Fbg are localized in the same structure in resting and thrombin-stimulated cells. On the ultrastructural level, an alpha granule localization for Fbg has previously been established.3-6 We have extended our immunofluorescent observations regarding the localization of Factor V in human platelets by use of transmission electron microscopy of antibody-stained ultrathin frozen sections. In resting cells, staining of virtually all alpha granules was observed for Factor V. In contrast, consistent staining was absent from other organelles including plasma membranes, mitochondria, and vacuolar structures which may represent the open canalicular system. These data thus establish at the ultrastructural level an alpha granule localization of human coagulation Factor V. © 1986 by Grune & Stratton, Inc.

Glanzmann thrombasthenia is an autosomal recessive disorder of the platelet glycoproteins (GP) IIb and IIIa. These glycoproteins normally serve as receptors for other adhesive glycoproteins, including fibrinogen, von Willebrand factor, and fibronectin. Most patients affected by Glanzmann thrombasthenia have low levels of GPIIb and GPIIIa; however, the separate mechanisms responsible for the deficiency in each remain to be determined. cDNA clones coding for the GPIIb and GPIIIa have been recently isolated, and their corresponding genomic sequences have been colocalized to the long arm of chromosome 17. Since a deletional event involving one or both of these structural genes could explain the disease phenotype, we have studied the DNA of two previously well-characterized cohorts of Glanzmann thrombasthenia patients from Israel. We performed Southern analysis with near full-length cDNA probes on genomic DNA obtained from 20 individuals. Four restriction enzyme digests were completed on each DNA sample. The similarity of banding patterns among probands, family members, and controls indicated that there were no major insertions or deletions in either the GPIIb or GPIIIa genes. Thus, the genetic defect in these patients with Glanzmann thrombasthenia is most likely due to either a small change in the nucleotide sequence of the coding region or a defect in the regulatory region of one or both genes.

Thrombospondin (TSP), a platelet a-granule protein, becomes expressed on the surface of thrombin-stimulated platelets. The surface expression of this protein occurs through two distinct mechanisms. At low platelet concen trations (1 × 108/mL), a divalent ion-independent, low-capacity mechanism predominates. At higher cell con centrations, a divalent ion-dependent, higher capacity mechanism prevails that can account for >90% of all the TSP surface expression measured. This mechanism requires the presence of both calcium and magnesium (Ca + Mg). The dependence of the divalent ion-dependent surface expression on platelet concentration suggests that release of the molecule from the cell followed by its binding to the cell surface mediates this component of the endoge nous TSP-platelet interaction. These data are consistent with a two-receptor model for the platelet surface expres sion of the endogenous TSP pool.

Platelet membrane GPIIb-IIIa is a member of the integrin family of heterodimeric adhesion receptors. Processing and export of certain leukocyte and melanoma integrins is disrupted in cells lacking one subunit. We found that surface expression of GPIIb-IIIa, measured by fluorescent activated cell sorting or by surface labeling, required cotransfection of both subunits. In contrast, surface expression was not detected when the subunits were transfected individually. Immunoprecipitation of metaboli- cally labeled transfected cells confirmed the presence of comparable levels of intracellular protein in all cases. When both subunits were transfected, post-translational cleavage of Pro-GPIIb to yield GPIIb heavy chain was also seen, while transfection with GPIIb alone resulted in coprecipitation of Pro-GPIIb with a second band that may be an endogenous β subunit. Pro-GPIIb in these transfectants was not processed to yield GPIIb heavy chain. When transfected into COS cells alone, transiently expressed GPIIIa remained intracellular and did not appear to complex with any endogenous proteins. Thus, surface expression of processed GPIIb-IIIa depends on the presence of both subunits; the coordinate reduction of both subunits observed in some cases of Glanzmann's thrombasthenia may result from mutation affecting only one.

We have measured thrombin-induced secretion of platelet factor 4 antigen (PF4) and simultaneously followed its intracellular translocation by immunofluorescence. In permeable resting platelets, speckled intracellular immunofluores-cent staining for PF4 was observed. Addition of thrombin to washed platelets at 22°C resulted in secretion of PF4 and formation of large (~0.5 μm) immunoflourescent masses. These masses moved to the cell periphery during secretion and were virtually absent at the conclusion of secretion. Ultrastructural examination of thrombin-treated platelets revealed vacuoles corresponding in size, shape, and time of occurrence to the large immunofluorescent masses of PF4. These vacuoles contained PF4 by immunoferritin staining of frozen thin sections; they therefore appear to represent the ultrastructural counterpart of the large PF4 masses. When intact cells were stained for PF4 after thrombin addition, only 5.6% of the large masses stained. Thus, during secretion, PF4 antigen is consolidated into large closed pools that appear as vacuoles in the electron microscope.

In Oz, the yellow brick road led to dreams come true. In platelets, the road from agonist receptors to aIIbß3 leads to thrombus formation, a response necessary for hemostasis and thrombosis. Upon platelet stimulation, agonist receptor pathways converge on the cytoplasmic and transmembrane domains of aIIbß3, causing conformational changes that are propagated to the extracellular domain to effect high affinity ligand binding (activation). Numerous intracellular proteins have been implicated inaIIbß3 activation, including protein kinase C (PKC), the Rap1 GTPase and talin. Indeed, talin recruitment to the ß3 cytoplasmic domain is a final common step in aIIbß3 activation. However, the relationships of these signaling proteins to each other, and the mechanism(s) of talin recruitment to ß3 have not been established. Here we used forward, reverse, and synthetic genetics to engineer and order an aIIbß3 activation pathway. In a CHO cell model system, increased expression of PKCaand talin to levels approximating those in platelets reconstituted aIIbß3 activation in response to phorbol myristate acetate (PMA). This response was detectable by monitoring fibrinogen binding or the binding of a monovalent, ligand-mimetic antibody Fab fragment (PAC-1 Fab) that is specific for high-affinity aIIbß3. PMA-induced activation of aIIbß3 could be blocked by over-expression of Rap1GAP, which inhibits Rap1 GTPase activity. In contrast, over-expression of constitutively-active Rap1A(G12V) caused aIIbß3 activation that bypassed the requirement for PKCa, indicating that Rap1 is downstream of PKCain this integrin activation pathway. aIIbß3 activation by Rap1 was promoted by a specific Rap1 effector, RIAM, an adapter molecule with a PH domain and proline-rich and EVH-1 binding regions. For example, over-expression of RIAM, but not another Rap1 effector (RapL), caused activation of aIIbß3 and bypassed the requirement for PKCaand Rap1. Furthermore, siRNA-mediated knockdown of RIAM blocked aIIbß3 activation by PMA or Rap1A(G12V), and this effect could be rescued by an siRNA-resistant form of RIAM. The integrin activating effects of PKCa, Rap1A(G12V) and RIAM were not observed if an integrin binding-defective talin mutant (W359A) was expressed instead of talin. In both CHO cells and platelets, activated Rap1 formed an integrin-associated complex with talin and RIAM, as assessed by deconvolution microscopy and co-immunoprecipitation. In addition, when RIAM was knocked down by siRNA in ES cell-derived murine megakaryocytes, aIIbß3 activation in response to ADP or thrombin receptor agonists was impaired. These results identify and order a signaling pathway from agonist stimulation to aIIbß3 activation. At the end of this road, Rap1 organizes an integrin activating complex composed of RIAM and talin. The ability to synthetically reconstruct an aIIbß3 activation pathway and validate it in platelets and megakaryocytes now provides molecular targets to facilitate characterization of clinical platelet activation defects and development of better anti-platelet drugs. We are not in Kansas anymore.