Mice Expressing Low Levels of CalDAG-GEFI Exhibit Markedly Impaired Platelet Activation With Minor Impact on Hemostasis

Abstract

Objective The tight regulation of platelet adhesiveness, mediated by the αIIbβ3 integrin, is critical for hemostasis and prevention of thrombosis. We recently demonstrated that integrin affinity in platelets is controlled by the guanine nucleotide exchange factor, CalDAG-GEFI (CD-GEFI), and its target, RAP1. In this study, we investigated whether low-level expression of CD-GEFI leads to protection from thrombosis without pathological bleeding in mice. Approach and Results Cdg1low mice were generated by knock-in of human CD-GEFI cDNA into the mouse Cdg1 locus. CD-GEFI expression in platelets from Cdg1low mice was reduced by ~90% when compared to controls. Activation of RAP1 and αIIbβ3 was abolished at low agonist concentrations and partially inhibited at high agonist concentrations in Cdg1low platelets. Consistently, the aggregation response of Cdg1low platelets was weaker than that of wild-type (WT) platelets, but more efficient than that observed in Cdg1−/− platelets. Importantly, Cdg1low mice were strongly protected from arterial and immune complex-mediated thrombosis, with only minimal impact on primary hemostasis. Conclusion Together, our studies suggest the partial inhibition of CD-GEFI function as a powerful new approach to safely prevent thrombotic complications.

Deletion of the Arp2/3 complex in megakaryocytes leads to microthrombocytopenia in mice

“…This marked reduction in Arp2/3 expression, however, has only a very mild effect on platelet integrin activation in vitro and hemostatic plug formation in vivo . In contrast, platelet adhesion, hemostasis and thrombosis were significantly impaired in mice with similar reductions in CalDAG-GEFI 37 or Kindlin-3 23 , highly expressed proteins critical to platelet adhesion and thrombus formation. Thus, the Arp2/3 complex does not seem to be a major regulator of hemostatic plug formation at sites of vascular damage.…”

Subcellular localization of Rap1 GTPase activator CalDAG‐GEFI is orchestrated by interaction of its atypical C1 domain with membrane phosphoinositides

“…The relatively mild bleeding defect in mice and adult human patients expressing CDGI lacking the C1 domain provides a rationale for targeting the C1‐phosphoinositide interaction in the prevention of atherothrombosis. Our studies in mice expressing CDGIΔC1 and mice expressing low levels of CDGI demonstrated strong protection from experimental thrombosis, including models of arterial thrombosis and immune complex‐mediated thrombocytopenia and thrombosis . On a mechanistic level, the protection from arterial thrombosis is most likely explained by a delay and reduction in integrin activation, the key event in platelet aggregation.…”

“…Our studies in mice expressing CDGIΔC1 and mice expressing low levels of CDGI demonstrated strong protection from experimental thrombosis, including models of arterial thrombosis and immune complex-mediated thrombocytopenia and thrombosis. 13,70 On a mechanistic level, the protection from arterial thrombosis is most likely explained by a delay and reduction in integrin activation, the key event in platelet aggregation. CDGI plays a critical role in the near-immediate activation of αIIbβ3, 8 with deficiency in the protein leading to markedly delayed platelet aggregation in mice and humans.…”

Losing your GRP on platelet activation

“…2 The importance of CalDAG GEFI in the intact vasculature has also been demonstrated in several mouse models of hemostasis, atherosclerosis, and thrombotic thrombocytopenia. [3][4][5] Even though human CalDAG GEFI is thought to perform the same function as its murine homolog, a glimpse of the physiologic role of CalDAG GEFI in human platelets has only recently been attained. A point mutation in the human RASGRP2 gene was discovered in 2 homozygous patients by wholeexome sequencing by Canault et al 6 These investigators showed that platelets expressing a CalDAG GEFI mutant (G248W) displayed decreased platelet spreading, aggregation, and adhesion under flow conditions without affecting aIIbb3 activation or CalDAG GEFI protein levels.…”