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Complement plays a key role in host defense, but its dysregulation can cause autologous tissue injury. Complement activation is normally controlled by regulatory proteins, including factor H (FH) in plasma and membrane cofactor protein (MCP) on the cell surface. Mutations in FH and MCP are linked to atypical hemolytic uremic syndrome, a type of thrombotic microangiopathy (TMA) that causes renal failure. We describe here that disruption of FH function on the cell surface can also lead to disseminated complement-dependent macrovascular thrombosis. By gene targeting, we introduced a point mutation (W1206R) into murine FH that impaired its interaction with host cells but did not affect its plasma complement-regulating activity. Homozygous mutant mice carrying this mutation developed renal TMA as well as systemic thrombophilia involving large blood vessels in multiple organs, including liver, lung, spleen, and kidney. Approximately 30% of mutant mice displayed symptoms of stroke and ischemic retinopathy, and 48% died prematurely. Genetic deficiency of complement C3 and factor D prevented both the systemic thrombophilia and renal TMA phenotypes. These results demonstrate a causal relationship between complement dysregulation and systemic angiopathy and suggest that complement activation may contribute to various human thrombotic disorders involving both the micro- and macrovasculature.

Platelet aggregation at sites of vascular injury is essential for hemostasis but also thrombosis. Platelet adhesiveness is critically dependent on agonist-induced inside-out activation of heterodimeric integrin receptors by a mechanism involving the recruitment of talin-1 to the cytoplasmic integrin tail. Experiments in heterologous cells have suggested a critical role of Rap1-guanosine triphosphate-interacting adaptor molecule (RIAM) for talin-1 recruitment and thus integrin activation, but direct in vivo evidence to support this has been missing. We generated RIAM-null mice and found that they are viable, fertile, and apparently healthy. Unexpectedly, platelets from these mice show unaltered β3- and β1-integrin activation and consequently normal adhesion and aggregation responses under static and flow conditions. Similarly, hemostasis and arterial thrombus formation were indistinguishable between wild-type and RIAM-null mice. These results reveal that RIAM is dispensable for integrin activation and function in mouse platelets, strongly suggesting the existence of alternative mechanisms of talin-1 recruitment.

The local microenvironment within an evolving hemostatic plug shapes the distribution of soluble platelet agonists, resulting in a gradient of platelet activation. We previously showed that thrombin activity at a site of vascular injury is spatially restricted, resulting in robust activation of a subpopulation of platelets in the hemostatic plug core. In contrast, adenosine 5'-diphosphate (ADP)/P2Y12 signaling contributes to the accumulation of partially activated, loosely packed platelets in a shell overlying the core. The contribution of the additional platelet agonists thromboxane A2 (TxA2) and epinephrine to this hierarchical organization was not previously shown. Using a combination of genetic and pharmacologic approaches coupled with real-time intravital imaging, we show that TxA2 signaling is critical and nonredundant with ADP/P2Y12 for platelet accumulation in the shell region but not required for full platelet activation in the hemostatic plug core, where thrombin activity is highest. In contrast, epinephrine signaling is dispensable even in the presence of a P2Y12 antagonist. Finally, dual P2Y12 and thrombin inhibition does not substantially inhibit hemostatic plug core formation any more than thrombin inhibition alone, providing further evidence that thrombin is the primary driver of platelet activation in this region. Taken together, these studies show for the first time how thrombin, P2Y12, and TxA2 signaling are coordinated during development of a hierarchical organization of hemostatic plugs in vivo and provide novel insights into the impact of dual antiplatelet therapy on hemostasis and thrombosis.

Most platelet agonists work through G protein coupled receptors (GPCRs), activating pathways that involve members of the Gq, Gi, and G12 families of heterotrimeric G proteins. Gq is critical for most functional responses during platelet activation. A defect in Gq expression in patients leads to impaired platelet secretion and aggregation, which is associated with bleeding diathesis. We have previously shown that the Regulators of G protein Signaling (RGS) proteins function as negative regulators for platelet activation by limiting G protein-dependent signaling, but much remains to be learned about the mechanisms by which G proteins can be regulated during platelet activation. Here we took advantage of an RGS-insensitive Gq(G188S) mutant mouse line to decipher the regulatory complex of Gq-dependent signaling in platelets. The G188S mutation disrupts RGS18 and Gq interaction in a way similar to what we have observed in an analogous Gi2(G184S) mutation. However, in contrast to increased platelet activation in Gi2(G184S) expressing platelets, G188S expressing platelets have decreased activation in response to thrombin, TxA2 and ADP, but not to collagen. The hemostatic thrombi formed in G188S mice are significantly reduced with constant embolization as compared with WT controls. Underlying these changes is a decrease in Gq-dependent signaling events, whereas shape change, which is G13-mediated, is unaffected. Our data further reveal that PLCβ3 and GRK2 complex with activated Gq, but not to Gi2. The G188S mutation in Gq not only prevents the binding of RGS proteins to Gq but also impairs the Gq/PLCβ3 interaction, though it does not affect the GRK2 binding to Gq. Instead, there an increase of GRK2 binding to Gq. Finally, the structural analysis shows that PLCβ3 shares a large overlapping binding area with RGS18 to Gq. Computational alanine scanning to predict binding interfaces indicates that the G188S mutation resides in a region important for the binding of both PLC and RGS. The PLC binding interface also overlaps with that of GRK2, which suggests that when PLC is in complex with Gq, it precludes the binding of both RGS proteins and GRK2. In the G188S mutant, however, PLC binding is reduced, which may reduce competition and allow more GRK2 to bind. The binding interfaces of RGS and GRK2 are not predicted to be overlapping, raising the possibility that the two proteins could bind simultaneously to activated WT Gq. Collectively, these observations 1) indicate that the feedback mechanism of Gq inactivation in platelets is different from that of Gi2, 2) reveal, for the first time, that a Gq/PLCβ/RGS/GRK2 signaling node is present in platelets and plays an important role in Gq-dependent signaling during platelet activation, and 3) show that the G188S mutation affect Gq-mediated signaling by disrupting Gq/PLCβ/RGS interaction. Disclosures No relevant conflicts of interest to declare.

The cellular and molecular mechanisms orchestrating the complex process by which bone marrow megakaryocytes form and release platelets remain poorly understood. Mature megakaryocytes generate long cytoplasmic extensions, proplatelets, which have the capacity to generate platelets. Although microtubules are the main structural component of proplatelets and microtubule sliding is known to drive proplatelet elongation, the role of actin dynamics in the process of platelet formation has remained elusive. Here, we tailored a mouse model lacking all ADF/n-cofilin–mediated actin dynamics in megakaryocytes to specifically elucidate the role of actin filament turnover in platelet formation. We demonstrate, for the first time, that in vivo actin filament turnover plays a critical role in the late stages of platelet formation from megakaryocytes and the proper sizing of platelets in the periphery. Our results provide the genetic proof that platelet production from megakaryocytes strictly requires dynamic changes in the actin cytoskeleton.

Wiskott-Aldrich syndrome (WAS) is a rare, X-chromosomal recessive disorder which is caused by mutations in the WAS gene and characterized by eczema, immunodeficiency and microthrombocytopenia (Thrasher and Burns, Nat Rev Immunol 2010). Interestingly, WAS protein (WASp)-deficient mice have normal-sized platelets and thus the molecular link between WAS mutations and its central hallmark microthrombocytopenia remains elusive. Profilin1 (Pfn1) is a key actin-regulating protein that, besides actin, interacts with phosphoinositides and multiple proline-rich proteins including the WAS protein (WASp)/WASp-interacting protein (WIP) complex (Witke, Trends Cell Biol 2004; Ramesh et al., Proc Natl Acad Sci USA, 1997). Interestingly, similar to WAS patients, mice with Pfn1-null megakaryocytes/platelets suffered from microthrombocytopenia. We identified accelerated platelet clearance by macrophages and pre-mature platelet release into the bone marrow compartment as the major cause of the reduced platelet count in Pfn1-deficient mice. Both, platelets from Pfn1-null mice and WAS patients contained abnormally organized and hyperstable microtubules. We next tested, if increased microtubule stability could account for the reduced size of Pfn1-deficient platelets. Treatment of control platelets with microtubule stabilizing toxins, such as the histone-deacetylase inhibitor trichostatin A (TSA) or taxol resulted in a decreased platelet size. This finding indicates that increased microtubule stability could account for the reduced platelet size in Pfn1-deficient mice but also in WAS patients. Based on these results we speculate that WASp might modulate Pfn1 function and dysregulation of this interaction leads to increased stability and altered organization of microtubules. In support of this, the subcellular localization of Pfn1 was altered in platelets of three WAS patients. Together, these results reveal an unexpected function of Pfn1 as a regulator of microtubule organization and point to a previously unrecognized mechanism underlying the platelet formation defect in WAS patients. Disclosures No relevant conflicts of interest to declare.