Rap1-GTP–interacting adaptor molecule (RIAM) is dispensable for platelet integrin activation and function in mice

Platelet adhesion and aggregation are essential for hemostasis; however, uncontrolled aggregation responses in injured vessels can cause life-threatening disease states such as myocardial infarction or stroke.^1,2  These processes are mediated by heterodimeric receptors of the β1- and β3-integrin families.³  In resting platelets, integrins are expressed in a low-affinity state, but in response to cellular activation they shift to a high-affinity state and efficiently bind their ligands, most notably components of the extracellular matrix and other receptors.^3-5  This so-called inside-out activation is triggered by the binding of talin-1 and kindlins to NPYX motifs at the intracellular tail of the integrin β-subunit.^6-8  The mechanisms of talin-1 recruitment to integrins have been extensively studied, and the small GTPase Rap1 and its downstream effector Rap1-guanosine triphosphate (GTP)-interacting adaptor molecule (RIAM) have been implicated therein.^9-12  RIAM, a member of the Mig-10/RIAM/lamellipodin family, contains a Ras-association and an adjacent pleckstrin homology domain, which suggests that it serves as a proximity detector for Rap1-GTP and phosphatidylinositol-4,5-bisphosphates.¹³  Thus, RIAM and bound talin-1 are located to membrane microdomains rich in phosphatidylinositol-4,5-bisphosphates, which has been shown to increase binding of talin-1 to the β-integrin subunit and ultimately lead to integrin activation.¹³  An important role of RIAM in platelet αIIbβ3-integrin activation has been demonstrated in different overexpression and knockdown studies in mammalian cell lines, but also in primary mouse megakaryocytes.^9-11,14  However, direct evidence of a role for RIAM in platelet integrin activation has not been provided.

Animal studies were approved by the district government of Lower Franconia (Bezirksregierung Unterfranken). Apbb1ip^+/− mice were generated by injection of embryonic stem cell clone 16066A-D5 (KOMP; supplemental Figure 1, available on the Blood Web site) into C57Bl/6 blastocysts. Germ-line transmission was obtained by backcrossing the resulting chimeric mice with C57Bl/6 mice.

Western blot analysis was performed as described previously.¹⁵  RIAM (1 μg mL⁻¹, EP2806; Epitomics) and β-actin (1 μg mL⁻¹, #4970; Cell Signaling) were probed with the respective antibodies.

Platelet count, size, and activation of β3- (JON/A-PE; Emfret) and β1-integrins (9EG7-FITC; BD Pharmingen), as well as surface exposure of α2-, α5-, β1-, αIIb-, and β3-integrins, were determined by flow cytometry as previously described.^6,7  Preparation of platelets, turbidometric aggregometry, and platelet spreading have been described elsewhere.^6,7,15,16 

Platelet clot retraction and adhesion on collagen under flow (1000 s⁻¹) were performed as described previously.^6,17 

Experimental procedures to assess hemostasis and arterial thrombosis are described elsewhere.^6,18 

The presented results are mean ± standard deviation from ≥3 independent experiments per group, if not otherwise stated. Differences between control and RIAM-null mice were statistically analyzed using the Student t test. P < .05 was considered statistically significant.

We targeted the Apbb1ip gene (supplemental Figure 1) by applying a VelociGene Definitive Null Allele Design, where exons 3 to 6 of the Apbb1ip gene were replaced with a LacZ-p(A); Neo-p(A) cassette by homologous recombination, thus abolishing Apbb1ip expression. Apbb1ip^+/− mice were intercrossed to obtain Apbb1ip^−/− (referred to as RIAM-null) and the respective control mice. RIAM-null mice were born at a normal Mendelian ratio, viable, and fertile, and appeared overall healthy. Western blot analysis confirmed strong expression of RIAM in control but not in RIAM-null platelets (Figure 1A). Platelet counts (Figure 1B), size (Figure 1C), and the distribution of red and white blood cells (data not shown) were indistinguishable from controls, suggesting that RIAM is not essential for hematopoiesis.

Flow cytometric analyses showed that control and RIAM-null platelets display comparable surface expression levels of β1- and β3-integrins both under resting conditions and upon stimulation with thrombin (Figure 1D). Next, agonist-induced αIIbβ3-integrin activation was determined using the JON/A-PE antibody¹⁹  and, remarkably, found to occur with the same efficiency in RIAM-null and wild-type platelets in response to all tested agonists (Figure 1E). Similar results were obtained for the binding of Alexa F488-conjugated fibrinogen (Figure 1F). These results were entirely unexpected given previous studies where a knockdown of RIAM was associated with impaired cell adhesion and αIIbβ3-integrin activation.^9-11  Of note, we also performed time course experiments for platelet fibrinogen binding and obtained comparable results at all tested time points for control and mutant platelets, strongly suggesting that integrin disengagement is not affected by RIAM deficiency (Figure 1F; data not shown). In agreement with these data, RIAM-null platelets displayed normal aggregation responses to different agonists (Figure 1G). Besides αIIbβ3-integrins, RIAM has been implicated in β1-integrin activation and adhesion of Jurkat T-cells.¹⁴  However, activation of β1-integrins, as assessed by binding of the 9EG7 antibody,²⁰  was comparable between RIAM-null and control platelets (Figure 1H). Consistently, RIAM-null platelets showed normal adhesion (Figure 1I) and aggregate formation (Figure 1J) on collagen in a flow adhesion assay, which is known to be highly dependent on functional α2β1 integrins.^6,7,21 

RIAM has recently been implicated in integrin outside-in signaling in melanoma cells.^22,23  To assess the role of RIAM in integrin outside-in signaling in platelets, we allowed control and RIAM-null platelets to spread on fibrinogen but found no differences in adhesion, extent, or kinetics of spreading (Figure 2A) or cytoskeletal rearrangements (Figure 2B). Likewise, integrin outside-in signaling-dependent clot retraction was indistinguishable between wild-type and RIAM-null mice (Figure 2C-D), excluding an essential role of RIAM in this process.

To investigate the role of RIAM in platelet integrin function in vivo, we used a tail bleeding time assay and found comparable bleeding times for control (7.2 ± 1.9 minutes) and RIAM-null mice (6.8 ± 2.1 minutes) (Figure 2E). Consistently, pathological thrombus formation, as assessed by intravital microscopy of FeCl₃-injured mesenteric arterioles, was indistinguishable between control and RIAM-null mice, resulting in similar occlusion times (16.4 ± 3.8 vs 15.3 ± 4.9 minutes, respectively) for both groups (Figure 2F-G).

Our results show that RIAM is dispensable for integrin inside-out and outside-in signaling in platelets and consequently for hemostasis and arterial thrombosis in mice. Other Mig-10/RIAM/lamellipodin family members might compensate for RIAM function; however, we and others¹²  were unable to detect lamellipodin expression in platelets, and Mig-10 is only found in Caenorhabditis elegans.^11,14  In contrast to results obtained in cell culture approaches,^9-11  talin-1 was normally recruited to β3-integrins in spread RIAM-null platelets (supplemental Figure 2). Consequently, talin-1 might be recruited to integrins via alternative pathways, eg, by interaction with the exocyst complex, or by binding to the focal adhesion kinase.^24,25  In support of this, Han and colleagues showed in CHO cells that talin-1 alone was sufficient to mediate β1- and β3-integrin activation, whereas RIAM and other Rap1 effectors were not.⁹  Moreover, because platelets lacking Rap1b or CalDAG-GEFI did not show a complete block of αIIbβ3-integrin activation, alternative mechanisms for platelet integrin activation beyond RIAM-mediated recruitment of talin-1 must be postulated.^12,21 

This work was supported by the Deutsche Forschungsgemeinschaft (SFB688 to B.N.). S.S. was supported by a grant of the German Excellence Initiative to the Graduate School of Life Sciences, University of Würzburg. The mouse strain used for this research project was created from ES cell clone 16066A-D5 obtained from the National Center for Research Resources (NCRR)-National Institutes of Health-supported KOMP Repository (www.komp.org) and generated by the CHORI/Sanger/UC Davis (CSD) consortium for the NIH-funded Knockout Mouse Project.

Contribution: S.S. performed experiments, analyzed data, and wrote the manuscript; K.W., V.L., T.V., S.G., and M.R.B. performed experiments and analyzed data; and B.N. supervised research, analyzed data, and wrote the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Bernhard Nieswandt, University Hospital and Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany; e-mail: bernhard.nieswandt@virchow.uni-wuerzburg.de.