Raymond Piatt

RASA3 is a critical inhibitor of RAP1-dependent platelet activation

The small GTPase RAP1 is critical for platelet activation and thrombus formation. RAP1 activity in platelets is controlled by the GEF CalDAG-GEFI and an unknown regulator that operates downstream of the adenosine diphosphate (ADP) receptor, P2Y12, a target of antithrombotic therapy. Here, we provide evidence that the GAP, RASA3, inhibits platelet activation and provides a link between P2Y12 and activation of the RAP1 signaling pathway. In mice, reduced expression of RASA3 led to premature platelet activation and markedly reduced the life span of circulating platelets. The increased platelet turnover and the resulting thrombocytopenia were reversed by concomitant deletion of the gene encoding CalDAG-GEFI. Rasa3 mutant platelets were hyperresponsive to agonist stimulation, both in vitro and in vivo. Moreover, activation of Rasa3 mutant platelets occurred independently of ADP feedback signaling and was insensitive to inhibitors of P2Y12 or PI3 kinase. Together, our results indicate that RASA3 ensures that circulating platelets remain quiescent by restraining CalDAG-GEFI/RAP1 signaling and suggest that P2Y12 signaling is required to inhibit RASA3 and enable sustained RAP1-dependent platelet activation and thrombus formation at sites of vascular injury. These findings provide insight into the antithrombotic effect of P2Y12 inhibitors and may lead to improved diagnosis and treatment of platelet-related disorders.

Novel mouse hemostasis model for real-time determination of bleeding time and hemostatic plug composition

Hemostasis is a rapid response by the body to stop bleeding at sites of vessel injury. Both platelets and fibrin are important for the formation of a hemostatic plug. Mice have been used to uncover the molecular mechanisms that regulate the activation of platelets and coagulation under physiologic conditions. However, measurements of hemostasis in mice are quite variable, and current methods do not quantify platelet adhesion or fibrin formation at the site of injury.

A talin mutant that impairs talin-integrin binding in platelets decelerates αIIbβ3 activation without pathological bleeding

Tight regulation of integrin affinity is critical for hemostasis. A final step of integrin activation is talin binding to 2 sites within the integrin β cytoplasmic domain. Binding of talin to a membrane-distal NPxY sequence facilitates a second, weaker interaction of talin with an integrin membrane-proximal region (MPR) that is critical for integrin activation. To test the functional significance of these distinct interactions on platelet function in vivo, we generated knock-in mice expressing talin1 mutants with impaired capacity to interact with the β3 integrin MPR (L325R) or NPLY sequence (W359A). Both talin1(L325R) and talin1(W359A) mice were protected from experimental thrombosis. Talin1(L325R) mice, but not talin(W359A) mice, exhibited a severe bleeding phenotype. Activation of αIIbβ3 was completely blocked in talin1(L325R) platelets, whereas activation was reduced by approximately 50% in talin1(W359A) platelets. Quantitative biochemical measurements detected talin1(W359A) binding to β3 integrin, albeit with a 2.9-fold lower affinity than wild-type talin1. The rate of αIIbβ3 activation was slower in talin1(W359A) platelets, which consequently delayed aggregation under static conditions and reduced thrombus formation under physiological flow conditions. Together our data indicate that reduction of talin-β3 integrin binding affinity results in decelerated αIIbβ3 integrin activation and protection from arterial thrombosis without pathological bleeding.

Chemoproteomic Discovery of AADACL1 as a Regulator of Human Platelet Activation

A comprehensive knowledge of the platelet proteome is necessary for understanding thrombosis and for envisioning antiplatelet therapies. To discover other biochemical pathways in human platelets, we screened platelets with a carbamate library designed to interrogate the serine hydrolase subproteome and used competitive activity-based protein profiling to map the targets of active carbamates. We identified an inhibitor that targets arylacetamide deacetylase-like 1 (AADACL1), a lipid deacetylase originally identified in invasive cancers. Using this compound, along with highly selective second-generation inhibitors of AADACL1, metabolomics, and RNA interference, we show that AADACL1 regulates platelet aggregation, thrombus growth, RAP1 and PKC activation, lipid metabolism, and fibrinogen binding to platelets and megakaryocytes. These data provide evidence that AADACL1 regulates platelet and megakaryocyte activation and highlight the value of this chemoproteomic strategy for target discovery in platelets.

CalDAG-GEFI Deficiency Reduces Atherosclerotic Lesion Development in Mice

Objective—Platelets are important for the development and progression of atherosclerotic lesions. However, relatively little is known about the contribution of platelet signaling to this pathological process. Our recent work identified 2 independent, yet synergistic, signaling pathways that lead to the activation of the small GTPase Rap1; one mediated by the guanine nucleotide exchange factor, CalDAG-GEFI (CDGI), the other by P2Y12, a platelet receptor for adenosine diphosphate and the target of antiplatelet drugs. In this study, we evaluated lesion formation in atherosclerosis-prone low-density lipoprotein receptor deficient (Ldlr−/−) mice lacking CDGI or P2Y12 in hematopoietic cells. Approach and Results—Lethally irradiated Ldlr−/− mice were reconstituted with bone marrow from wild-type (WT), Caldaggef1−/− (cdgI−/−), p2y12−/−, or cdgI−/−p2y12−/− (double knockout [DKO]) mice and fed a high-fat diet for 12 weeks. Ldlr−/− chimeras deficient for CDGI or P2Y12 developed significantly smaller atherosclerotic lesions in the aortic sinus and in aortas when compared with the Ldlr−/−/WT controls. We also observed a significant reduction in platelet-leukocyte aggregates in blood from hypercholesterolemic Ldlr−/−/cdgI−/− and Ldlr−/−/p2y12−/− chimeras. Consistently, fewer macrophages and neutrophils were detected in the aortic sinus of Ldlr−/−/cdgI−/− and Ldlr−/−/ p2y12−/− chimeras. Compared with controls, the plaque collagen content was significantly higher in Ldlr−/− chimeras lacking CDGI. Interestingly, no statistically significant additive effects were seen in Ldlr−/−/DKO chimeras when compared with chimeras lacking only CDGI. Conclusions—Our findings suggest that CDGI is critical for atherosclerotic plaque development in hypercholesterolemic Ldlr−/− mice because of its contribution to platelet-leukocyte aggregate formation and leukocyte recruitment to the lesion area.

The Phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3)-binder Rasa3 Regulates Phosphoinositide 3-kinase (PI 3-kinase)-dependent Integrin αIIbβ3 Outside-in Signaling

The class I PI3K family of lipid kinases plays an important role in integrin αIIbβ3 function, thereby supporting thrombus growth and consolidation. Here, we identify Ras/Rap1GAP Rasa3 (GAP1IP4BP) as a major phosphatidylinositol 3,4,5-trisphosphate-binding protein in human platelets and a key regulator of integrin αIIbβ3 outside-in signaling. We demonstrate that cytosolic Rasa3 translocates to the plasma membrane in a PI3K-dependent manner upon activation of human platelets. Expression of wild-type Rasa3 in integrin αIIbβ3-expressing CHO cells blocked Rap1 activity and integrin αIIbβ3-mediated spreading on fibrinogen. In contrast, Rap1GAP-deficient (P489V) and Ras/Rap1GAP-deficient (R371Q) Rasa3 had no effect. We furthermore show that two Rasa3 mutants (H794L and G125V), which are expressed in different mouse models of thrombocytopenia, lack both Ras and Rap1GAP activity and do not affect integrin αIIbβ3-mediated spreading of CHO cells on fibrinogen. Platelets from thrombocytopenic mice expressing GAP-deficient Rasa3 (H794L) show increased spreading on fibrinogen, which in contrast to wild-type platelets is insensitive to PI3K inhibitors. Together, these results support an important role for Rasa3 in PI3K-dependent integrin αIIbβ3-mediated outside-in signaling and cell spreading.

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

Objective—The tight regulation of platelet adhesiveness, mediated by the &agr;IIb&bgr;3 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 knockin of human CD-GEFI cDNA into the mouse Cdg1 locus. CD-GEFI expression in platelets from Cdg1low mice was reduced by ≈90% when compared with controls. Activation of RAP1 and &agr;IIb&bgr;3 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 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. Conclusions—Together, our studies suggest the partial inhibition of CD-GEFI function as a powerful new approach to safely prevent thrombotic complications.

Effects of ibrutinib treatment on murine platelet function during inflammation and in primary hemostasis

Ibrutinib is an irreversible inhibitor of Bruton’s tyrosine kinase (BTK), which has emerged as a potent molecular therapy in the treatment of B-cell malignancies, and continues to be investigated for treatment in a variety of blood cancers. Despite an excellent safety profile and high tolerability

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

Actin reorganization regulates key processes in platelet activation. Here we examined the role of the Arp2/3 complex, an essential component in actin filament branching, in platelet function. The Arpc2 gene, encoding the p34 subunit of the Arp2/3 complex, was deleted in the megakaryocyte lineage (Arpc2fl/flPF4-Cre). Deletion of the Arp2/3 complex resulted in marked microthrombocytopenia in mice, caused by premature platelet release into the bone marrow compartment and impaired platelet survival in circulation. Arpc2fl/flPF4-Cre platelets exhibited alterations in their actin cytoskeleton and their peripheral microtubule coil. Thrombocytopenia was alleviated following clodronate liposome-induced macrophage depletion in Arpc2fl/flPF4-Cre mice. Arpc2fl/flPF4-Cre platelets failed to spread and showed a mild defect in integrin activation and aggregation. However, no significant differences in hemostasis or thrombosis were observed between Arpc2fl/flPF4-Cre and control mice. Thus, Arp2/3 is critical for platelet homeostasis but plays only a minor role for vascular hemostasis.

Small molecule targeting the Rac1-NOX2 interaction prevents collagen-related peptide and thrombin-induced reactive oxygen species generation and platelet activation

Essentials Reactive oxygen species (ROS) generation by NOX2 plays a critical role in platelet activation. Rac1 regulation of NOX2 is important for ROS generation. Small molecule inhibitor of the Rac1‐p67phox interaction prevents platelet activation. Pharmacologic targeting of Rac1‐NOX2 axis can be a viable approach for antithrombotic therapy.