Owen J. T. McCarty

GPVI and integrin alphaIIb beta3 signaling in platelets.

Summary.  This review summarizes recent developments in our understanding of the molecular basis of platelet activation by two distinct types of surface receptor, the immunoglobulin GPVI, and the integrin αIIbβ3 (also known as GPIIbIIIa). These two classes of receptor signal through similar yet distinct tyrosine kinase‐based signaling cascades leading to activation of phospholipase C γ2. The significance of these signaling cascades in platelet adhesion and platelet aggregation at arterial rates of shear is discussed.

Rac1 Is Essential for Platelet Lamellipodia Formation and Aggregate Stability under Flow

The role of Rac family proteins in platelet spreading on matrix proteins under static and flow conditions has been investigated by using Rac-deficient platelets. Murine platelets form filopodia and undergo limited spreading on fibrinogen independent of Rac1 and Rac2. In the presence of thrombin, marked lamellipodia formation is observed on fibrinogen, which is abrogated in the absence of Rac1. However, Rac1 is not required for thrombin-induced aggregation or elevation of F-actin levels. Formation of lamellipodia on collagen and laminin is also Rac1-dependent. Analysis of platelet adhesion dynamics on collagen under flow conditions in vitro revealed that Rac1 is required for platelet aggregate stability at arterial rates of shear, as evidenced by a dramatic increase in platelet embolization. Furthermore, studies employing intravital microscopy demonstrated that Rac1 plays a critical role in the development of stable thrombi at sites of vascular injury in vivo. Thus, our data demonstrated that Rac1 is essential for lamellipodia formation in platelets and indicated that Rac1 is required for aggregate integrity leading to thrombus formation under physiologically relevant levels of shear both in vitro and in vivo.

GPVI and integrin αIIbβ3 signaling in platelets

Summary.  This review summarizes recent developments in our understanding of the molecular basis of platelet activation by two distinct types of surface receptor, the immunoglobulin GPVI, and the integrin αIIbβ3 (also known as GPIIbIIIa). These two classes of receptor signal through similar yet distinct tyrosine kinase‐based signaling cascades leading to activation of phospholipase C γ2. The significance of these signaling cascades in platelet adhesion and platelet aggregation at arterial rates of shear is discussed.

A role for factor XIIa–mediated factor XI activation in thrombus formation in vivo

Mice lacking factor XII (fXII) or factor XI (fXI) are resistant to experimentally-induced thrombosis, suggesting fXIIa activation of fXI contributes to thrombus formation in vivo. It is not clear whether this reaction has relevance for thrombosis in pri mates. In 2 carotid artery injury models (FeCl(3) and Rose Bengal/laser), fXII-deficient mice are more resistant to thrombosis than fXI- or factor IX (fIX)-deficient mice, raising the possibility that fXII and fXI function in distinct pathways. Antibody 14E11 binds fXI from a variety of mammals and interferes with fXI activation by fXIIa in vitro. In mice, 14E11 prevented arterial occlusion induced by FeCl(3) to a similar degree to total fXI deficiency. 14E11 also had a modest beneficial effect in a tissue factor-induced pulmonary embolism model, indicating fXI and fXII contribute to thrombus formation even when factor VIIa/tissue factor initiates thrombosis. In baboons, 14E11 reduced platelet-rich thrombus growth in collagen-coated grafts inserted into an arteriovenous shunt. These data support the hypothesis that fXIIa-mediated fXI activation contributes to thrombus formation in rodents and primates. Since fXII deficiency does not impair hemostasis, targeted inhibition of fXI activation by fXIIa may be a useful antithrombotic strategy associated with a low risk of bleeding complications.

Prevention of vascular graft occlusion and thrombus-associated thrombin generation by inhibition of factor XI

The protease thrombin is required for normal hemostasis and pathologic thrombogenesis. Since the mechanism of coagulation factor XI (FXI)-dependent thrombus growth remains unclear, we investigated the contribution of FXI to thrombus formation in a primate thrombosis model. Pretreatment of baboons with a novel anti-human FXI monoclonal antibody (aXIMab; 2 mg/kg) inhibited plasma FXI by at least 99% for 10 days, and suppressed thrombin-antithrombin (TAT) complex and beta-thromboglobulin (betaTG) formation measured immediately downstream from thrombi forming within collagen-coated vascular grafts. FXI inhibition with aXIMab limited platelet and fibrin deposition in 4-mm diameter grafts without an apparent increase in D-dimer release from thrombi, and prevented the occlusion of 2-mm diameter grafts without affecting template bleeding times. In comparison, pretreatment with aspirin (32 mg/kg) prolonged bleeding times but failed to prevent graft occlusion, supporting the concept that FXI blockade may offer therapeutic advantages over other antithrombotic agents in terms of bleeding complications. In whole blood, aXIMab prevented fibrin formation in a collagen-coated flow chamber, independent of factor XII and factor VII. These data suggest that endogenous FXI contributes to arterial thrombus propagation through a striking amplification of thrombin generation at the thrombus luminal surface.

Factor XII inhibition reduces thrombus formation in a primate thrombosis model

The plasma zymogens factor XII (fXII) and factor XI (fXI) contribute to thrombosis in a variety of mouse models. These proteins serve a limited role in hemostasis, suggesting that antithrombotic therapies targeting them may be associated with low bleeding risks. Although there is substantial epidemiologic evidence supporting a role for fXI in human thrombosis, the situation is not as clear for fXII. We generated monoclonal antibodies (9A2 and 15H8) against the human fXII heavy chain that interfere with fXII conversion to the protease factor XIIa (fXIIa). The anti-fXII antibodies were tested in models in which anti-fXI antibodies are known to have antithrombotic effects. Both anti-fXII antibodies reduced fibrin formation in human blood perfused through collagen-coated tubes. fXII-deficient mice are resistant to ferric chloride-induced arterial thrombosis, and this resistance can be reversed by infusion of human fXII. 9A2 partially blocks, and 15H8 completely blocks, the prothrombotic effect of fXII in this model. 15H8 prolonged the activated partial thromboplastin time of baboon and human plasmas. 15H8 reduced fibrin formation in collagen-coated vascular grafts inserted into arteriovenous shunts in baboons, and reduced fibrin and platelet accumulation downstream of the graft. These findings support a role for fXII in thrombus formation in primates.

Activated protein C ligation of ApoER2 (LRP8) causes Dab1-dependent signaling in U937 cells

Binding of activated protein C (APC) to cells triggers multiple beneficial cytoprotective activities that suppress apoptosis, inflammation, and endothelial barrier breakdown. One paradigm for APCs signaling emphasizes its binding to endothelial cell protein C receptor (EPCR) and subsequent protease activated receptor (PAR)-1 activation. Here we used human monocytic-like U937 cells to evaluate apolipoprotein E receptor 2 (ApoER2)-dependent signaling by APC and found that APC initiated rapid phosphorylation of Tyr-220 in the adaptor protein disabled-1 (Dab1) and of Ser-473 in Akt. APC also induced phosphorylation of Ser-9 in glycogen synthase kinase 3β (GSK3β), which was blocked by the PI3K inhibitor LY294002. Receptor-associated protein (RAP), a general antagonist for binding of ligands to LDL receptor family members, inhibited APC-induced phosphorylation of Dab1 and GSK3β, whereas anti-EPCR or anti-PAR1 blocking antibodies did not. Knocking down ApoER2 by using siRNA-ablated APC induced Dab1 phosphorylation, suggesting that RAP-sensitive APC-induced signaling requires ApoER2. In surface plasmon resonance equilibrium binding studies, APC bound with high affinity to soluble (s) ApoER2 (apparent Kd, ≈30 nM) but not to soluble very low density lipoprotein receptor. RAP blocked APC binding to sApoER2 but not to sEPCR. RAP blocked binding of U937 cells to immobilized APC. RAP also blocked APCs ability to inhibit endotoxin-induced tissue factor pro-coagulant activity of U937 cells. Thus, we propose that ligation of ApoER2 by APC signals via Dab1 phosphorylation and subsequent activation of PI3K and Akt and inactivation of GSK3β, thereby contributing to APCs beneficial effects on cells.

Rho GTPases in Platelet Function

Summary.  The Rho family of GTP binding proteins, also commonly referred to as the Rho GTPases, are master regulators of the platelet cytoskeleton and platelet function. These low‐molecular‐weight or ‘small’ GTPases act as signaling switches in the spatial and temporal transduction, and amplification of signals from platelet cell surface receptors to the intracellular signaling pathways that drive platelet function. The Rho GTPase family members RhoA, Cdc42 and Rac1 have emerged as key regulators in the dynamics of the actin cytoskeleton in platelets and play key roles in platelet aggregation, secretion, spreading and thrombus formation. Rho GTPase regulators, including GEFs and GAPs and downstream effectors, such as the WASPs, formins and PAKs, may also regulate platelet activation and function. In this review, we provide an overview of Rho GTPase signaling in platelet physiology. Previous studies of Rho GTPases and platelets have had a shared history, as platelets have served as an ideal, non‐transformed cellular model to characterize Rho function. Likewise, recent studies of the cell biology of Rho GTPase family members have helped to build an understanding of the molecular regulation of platelet function and will continue to do so through the further characterization of Rho GTPases as well as Rho GAPs, GEFs, RhoGDIs and Rho effectors in actin reorganization and other Rho‐driven cellular processes.

Evaluation of the role of platelet integrins in fibronectin‐dependent spreading and adhesion

Summary.  Background: Recent studies have shown that platelet adhesion and subsequent aggregation can occur in vivo in the absence of the two principal platelets adhesive ligands, von Willebrand factor and fibrinogen. These results highlight a possible role for fibronectin in supporting thrombus formation. Objective and methods: To evaluate the platelet integrins and subsequent activation pathways associated with fibronectin‐dependent platelet adhesion utilizing both human and murine platelets. Results: Platelets can adhere to fibronectin via the integrin αIIbβ3, leading to formation of lamellipodia. This is mediated through an interaction with the tenth type III domain in fibronectin. Spreading on fibronectin promotes αIIbβ3‐mediated Ca2+ mobilization and tyrosine phosphorylation of focal adhesion kinase and phospholipase C γ2. In contrast, studies with blocking antibodies and mice demonstrate that α5β1 and αvβ3 support adhesion and promote formation of filopodia but not lamellipodia or tyrosine phosphorylation of these proteins. Further, neither α5β1 nor αvβ3 is able to induce formation of lamellipodia in the presence of platelets agonists, such as collagen‐related‐peptide (CRP). Conclusions: These observations demonstrate that integrins α5β1 and αvβ3 support platelet adhesion and the generation of filopodia but that, in contrast to the integrin αIIbβ3, are unable to promote formation of lamellipodia.

Microfluidics and Coagulation Biology

The study of blood ex vivo can occur in closed or open systems, with or without flow. Microfluidic devices, which constrain fluids to a small (typically submillimeter) scale, facilitate analysis of platelet function, coagulation biology, cellular biorheology, adhesion dynamics, and pharmacology and, as a result, can be an invaluable tool for clinical diagnostics. An experimental session can accommodate hundreds to thousands of unique clotting, or thrombotic, events. Using microfluidics, thrombotic events can be studied on defined surfaces of biopolymers, matrix proteins, and tissue factor, under constant flow rate or constant pressure drop conditions. Distinct shear rates can be generated on a device using a single perfusion pump. Microfluidics facilitated both the determination of intraluminal thrombus permeability and the discovery that platelet contractility can be activated by a sudden decrease in flow. Microfluidic devices are ideal for multicolor imaging of platelets, fibrin, and phosphatidylserine and provide a human blood analog to mouse injury models. Overall, microfluidic advances offer many opportunities for research, drug testing under relevant hemodynamic conditions, and clinical diagnostics.