Rap1b is required for normal platelet function and hemostasis in mice

Abstract

Rap1b, an abundant small GTPase in platelets, becomes rapidly activated upon stimulation with agonists. Though it has been implicated to act downstream from G protein-coupled receptors (GPCRs) and upstream of integrin alpha IIbbeta3, the precise role of Rap1b in platelet function has been elusive. Here we report the generation of a murine rap1b knockout and show that Rap1b deficiency results in a bleeding defect due to defective platelet function. Aggregation of Rap1b-null platelets is reduced in response to stimulation with both GPCR-linked and GPCR-independent agonists. Underlying the defective Rap1b-null platelet function is decreased activation of integrin alphaIIbbeta3 in response to stimulation with agonists and signaling downstream from the integrin alpha IIbbeta3. In vivo, Rap1b-null mice are protected from arterial thrombosis. These data provide genetic evidence that Rap1b is involved in a common pathway of integrin activation, is required for normal hemostasis in vivo, and may be a clinically relevant antithrombotic therapy target.

Figure 1

Targeted inactivation of the rap1b gene. (A) The murine rap1b gene consists of 6 coding (bands) and 1 untranslated exon (3′ UTR, open box). (B) The targeting vector contains 7.8 kb of genomic DNA flanking the neomycin-resistance cassette (neo). TK, thymidine kinase. (C) After homologous recombination, the neo cassette replaces the complete coding sequence of the rap1b gene. (D) Southern blot analysis of mouse tail DNA from heterozygous intercrosses digested with SspI and KpnI using a probe (P) that detects 5.5-kb and 9-kb fragments in the wild-type and knockout allele, respectively. (E) Western blot analysis of protein expression in platelets of indicated genotype. (F) Morphology of E15.5 wild-type (+/+) and Rap1b-null (–/–) embryos. Scale bar: 1 mm. E, EcoRI; H, HinDIII; K, KpnI; S, SspI.

Figure 2

Prolonged tail bleeding time in Rap1b-null mice and in Rap1b-null bone marrow chimeras. Plotted are data from normal mice and bone marrow recipients with normal platelet counts in which the appropriate phenotype of the platelets was confirmed by Western blotting analysis. For each data set, the box shows the 25th to 75th percentile range and median, which in the KO group overlaps with the 75th percentile. Whiskers extend to the 5th and 95th percentiles and mean and SEM are plotted to the right of the box. WT/WT, chimeric normal mice transplanted with normal bone marrow; KO/WT, chimeric normal mice transplanted with Rap1b-null bone marrow.

Figure 3

Inhibition of aggregation responses in Rap1b-null platelets. Typical aggregation traces of wild-type (+/+, black lines) and Rap1b-null (–/–, red lines) washed platelets in response to the following agonists: (A) ADP, (B) epinephrine, (C) type-I collagen, (D) convulxin, (E) AYPGKF peptide, and (F) calcium ionophore A23187. Arrows indicate 50% light transmission. Graphs represent the average maximum (max.) aggregation of rap1b-null platelets relative to that of normal platelets at a given agonist concentration. Error bars represent SD (n ≥ 4).

Figure 4

Rap1b-null platelets are susceptible to inhibition by purinergic receptor antagonists and cAMP. Typical aggregation traces of wild-type (+/+, black lines) and Rap1b-null (–/–, red lines) washed platelets and PRP in response to agonists in the absence and presence of indicated inhibitors. (A) PRP response to 5 μM ADP. (B) Aggregation response of washed platelets to 50 μg/ml collagen. (C) Aggregation response of washed platelets to 0.1 mM AYPGKF peptide. Arrows indicate 50% light transmission. n = 3. 2-MeSAMP, 2-methylthio-AMP; A3P5P, adenosine 3′-5′-diphosphate; cBIMPS, Sp-5,6-DCL-cBIMPS.

Figure 5

Impaired soluble and solid-phase fibrinogen binding to Rap1b-null platelets. (A) Specific soluble fibrinogen binding to Rap1b-null platelets, presented relative to that of normal platelets, arbitrarily set at 100%, in the presence of the indicated concentrations of ADP. (B) Specific soluble fibrinogen binding to Rap1b-null platelets, relative to that of normal platelets, in the presence of AYPGKF peptide. (C) Wild-type and Rap1b-null platelet spreading on fibrinogen-coated coverslips in the absence (FGN) or presence (FGN + epi) of epinephrine. Data represent mean ± SEM (n > 30). P < 0.0001 by t test compared with control.

Figure 6

Inhibition of arterial thrombus formation in rap1b-deficient mice. Blood flow through the carotid artery monitored immediately before and after FeCl₃ injury to the artery, represented as time 0. (A) Blood flow trace of a wild-type mouse. (B) Blood flow trace of a Rap1b-null mouse. Time of occlusion in the control mice was 7.0 ± 1.30 minutes (mean ± SD; n = 6). The blood flow did not stop in the Rap1b-null mice throughout the time of experiment (n = 6).