The Modifier of hemostasis (Mh) locus on chromosome 4 controls in vivo hemostasis of Gp6−/− mice

Abstract

Platelet glycoprotein VI (GPVI) is a key receptor for collagens that mediates the propagation of platelet attachment and activation. Targeted disruption of the murine gene Gp6 on a mixed 129 ؋ 1/ SvJ ؋ C57BL/6J background causes the expected defects in collagen-dependent platelet responses in vitro. The extent of this dysfunction in all Gp6 ؊/؊ mice is uniform and is not affected by genetic background. However, the same Gp6 ؊/؊ mice exhibit 2 diametrically opposed phenotypes in vivo. In some mice, tail bleeding times are extremely prolonged, and thrombus formation in an in vivo carotid artery ferric chloride-injury model is significantly impaired. In other littermates, tail bleeding times are within the range of wild-type mice, and in vivo thrombus formation is indistinguishable from that of control mice. Directed intercrosses revealed that these phenotypes are heritable, and a genome-wide singlenucleotide polymorphism scan revealed the most significant linkage to a single locus (8 megabases) on chromosome 4 (logarithm of the odds [LOD] score ‫؍‬ 6.9, P < .0001) that we designate Modifier of hemostasis (Mh) . IntroductionGlycoprotein VI (GPVI), which is restricted in expression to megakaryocytes and platelets, is an important receptor for collagens. GPVI (60-65 kDa) is noncovalently associated with the fragment crystallizable receptor ␥ (FcR␥) subunit, an immunoreceptor tyrosine-based activation motif (ITAM)-based signaling coreceptor, 1,2 and in the mouse, surface GPVI expression requires coexpression of FcR␥. 3 Upon engagement of collagens, the GPVI-FcR␥ complex transduces outside-in signals that involve spleen tyrosine kinase (Syk), linker for the activation of T cells (LAT), and Src homology 2 domain-containing leukocyte-specific phosphoprotein of 76 kDa (SLP-76), resulting in phospholipase ␥2 and phosphatidylinositol 3-kinase activation, leading to release of granule contents, prothrombinase activity, and platelet aggregation. [4][5][6][7] Targeted gene deletion in the mouse is one strategy to generate experimental models to assess the role of membrane receptors in hemostasis and thrombosis. We were the first to report the targeted disruption of murine Gp6 8 and demonstrated, using functional in vitro assays, that platelets from homozygous Gp6 Ϫ/Ϫ mice fail to aggregate upon stimulation with type I fibrillar collagen and fail to form thrombi upon perfusion over collagen-coated surfaces. However, the in vivo tail bleeding times for the same Gp6 Ϫ/Ϫ mice were more often within the range seen with wild-type littermates (45-140 seconds), and only a minority of the Gp6 Ϫ/Ϫ F2 littermates (3/13) exhibited an abnormal, extremely prolonged tail bleeding time (Ͼ 600 seconds). Similar proportions of normal and abnormal mice have been observed in studies of genetically engineered FcR␥ Ϫ/Ϫ mice, in which platelet surface GPVI expression is ablated, or wild-type mice made GPVI-deficient after 7 days by intraperitoneal injection of rat anti-mouse GPVI monoclonal antibody JAQ1. [9][10][11] On repeated examination, we dete...

Redundancy and Interaction of Thrombin- and Collagen-Mediated Platelet Activation in Tail Bleeding and Carotid Thrombosis in Mice

“…These results are consistent with at least some published studies (see Discussion) in which Par4 and GPVI mutants were studied separately 16–18,26,29 . Thus, in contrast to thrombin-signaling, GPVI signaling was unnecessary for normal thrombus formation when other platelet signaling pathways were left intact.…”

“…Some variability might be due to different means of achieving GPVI loss of function, e.g., FcRγ mutation and antibody-induced GPVI shedding might have effects beyond loss of GPVI signaling. Variability across studies of Gp6 nulls may also be due in part to different genetic backgrounds 26 . The Gp6 and Par mutant mice examined in this study had been backcrossed >7 times into a C57BL6 background and littermates were compared.…”

α2β1 Integrin, GPVI Receptor, and Common FcRγ Chain on Mouse Platelets Mediate Distinct Responses to Collagen in Models of Thrombosis

“…A study by Cheli et al [25] using the same mixed GPVI −/− mice on the 129/SvJ x C57BL/6 background identified a Modifier of hemostasis ( Mh ) locus on chromosome 4 that correlated with the extreme but transient dichotomy in tail bleeding time (tBT) seen in the earlier generations of these mice. A modest correlation was also observed in the in vivo ferric chloride-induced carotid artery injury model.…”

Distinct spatio-temporal Ca2+ signaling elicited by integrin α2β1 and glycoprotein VI under flow

“…1 Moreover, evidence is emerging that the function of collagen and/or collagen receptors on platelets may be subjected to the modifying effects of still unknown gene products. 41 Here, to highlight the role of ␣2␤1, we have used acid-soluble collagen type I, which does not exist as such in vivo but is composed of an assembly of fibrils in a helical configuration that may mimic the properties of the spiraled collagens identified in normal and pathologic tissues. 42 To extend the significance of our findings, we have also used collagen type VI, which platelets contact directly when the subendothelial matrix is exposed or, associated with collagen type I and III, when lesions reach deeper layers of the vessel wall.…”