Streaming potential in bio-mimetic microvessels mediated by capillary glycocalyx.

Implantable medical devices and biosensors are pivotal in revolutionizing the field of medical technology by opening new dimensions in the field of disease detection and cure. These devices need to harness a biocompatible and physiologically sustainable safe power source instead of relying on external stimuli, overcoming the constraints on their applicability in-vivo. Here, by appealing to the interplay of electromechanics and hydrodynamics in physiologically relevant microvessels, we bring out the role of charged endothelial glycocalyx layer (EGL) towards establishing a streaming potential across physiological fluidic conduits. We account for the complex rheology of blood-mimicking fluid by appealing to Newtonian fluid model representing the blood plasma and a viscoelastic fluid model representing the whole blood. We model the EGL as a poroelastic layer with volumetric charge distribution. Our results reveal that for physiologically relevant micro-flows, the streaming potential induced is typically of the order of 0.1 V/mm, which may turn out to be substantial towards energizing biosensors and implantable medical devices whose power requirements are typically in the range of micro to milliwatts. We also bring out the specific implications of the relevant physiological parameters towards establishment of the streaming potential, with a vision of augmenting the same within plausible functional limits. We further unveil that the dependence of streaming potential on EGL thickness might be one of the key aspects in unlocking the mystery behind the angiogenesis pattern. Our results may open up novel bio-sensing and actuating possibilities in medical diagnostics as well as may provide a possible alternative regarding the development of physiologically safe and biocompatible power sources within the human body.

GPIbα is required for platelet-mediated hepatic thrombopoietin generation.

Thrombopoietin (TPO), a hematopoietic growth factor produced predominantly by the liver, is essential for thrombopoiesis. Prevailing theory posits that circulating TPO levels are maintained through its clearance by platelets and megakaryocytes via surface c-Mpl receptor internalization. Interestingly, we found a two- to threefold decrease in circulating TPO in GPIbα-/- mice compared with wild-type (WT) controls, which was consistent in GPIbα-deficient human Bernard-Soulier syndrome (BSS) patients. We showed that lower TPO levels in GPIbα-deficient conditions were not due to increased TPO clearance by GPIbα-/- platelets but rather to decreased hepatic TPO mRNA transcription and production. We found that WT, but not GPIbα-/-, platelet transfusions rescued hepatic TPO mRNA and circulating TPO levels in GPIbα-/- mice. In vitro hepatocyte cocultures with platelets or GPIbα-coupled beads further confirm the disruption of platelet-mediated hepatic TPO generation in the absence of GPIbα. Treatment of GPIbα-/- platelets with neuraminidase caused significant desialylation; however, strikingly, desialylated GPIbα-/- platelets could not rescue impaired hepatic TPO production in vivo or in vitro, suggesting that GPIbα, independent of platelet desialylation, is a prerequisite for hepatic TPO generation. Additionally, impaired hepatic TPO production was recapitulated in interleukin-4/GPIbα-transgenic mice, as well as with antibodies targeting the extracellular portion of GPIbα, demonstrating that the N terminus of GPIbα is required for platelet-mediated hepatic TPO generation. These findings reveal a novel nonredundant regulatory role for platelets in hepatic TPO homeostasis, which improves our understanding of constitutive TPO regulation and has important implications in diseases related to GPIbα, such as BSS and auto- and alloimmune-mediated thrombocytopenias.

Selective Tropism of Dengue Virus for Human Glycoprotein Ib.

Since the hemorrhage in severe dengue seems to be primarily related to the defect of the platelet, the possibility that dengue virus (DENV) is selectively tropic for one of its surface receptors was investigated. Flow cytometric data of DENV-infected megakaryocytic cell line superficially expressing human glycoprotein Ib (CD42b) and glycoprotein IIb/IIIa (CD41 and CD41a) were analyzed by our custom-written software in MATLAB. In two-dimensional analyses, intracellular DENV was detected in CD42b+, CD41+ and CD41a+ cells. In three-dimensional analyses, the DENV was exclusively detected in CD42b+ cells but not in CD42b- cells regardless of the other expressions. In single-cell virus-protein analyses, the amount of DENV was directly correlated with those of CD42b at the Pearson correlation coefficient of 0.9. Moreover, RT- PCR and apoptosis assays showed that DENV was able to replicate itself and release its new progeny from the infected CD42b+ cells and eventually killed those cells. These results provide evidence for the involvement of CD42b in DENV infection.

The Candidate Schizophrenia Risk Gene DGCR2 Regulates Early Steps of Corticogenesis.

BACKGROUND: Alterations in early steps of cortical circuit assembly are thought to play a critical role in vulnerability to schizophrenia (SZ), but the pathogenic impact of SZ-risk mutations on corticogenesis remains to be determined. DiGeorge syndrome critical region 2 (DGCR2) is located in the 22q11.2 locus, whose deletion is a major risk factor for SZ. Moreover, exome sequencing of individuals with idiopathic SZ identified a rare missense mutation in DGCR2, further suggesting that DGCR2 is involved in SZ. METHODS: Here we investigated the function of Dgcr2 and the pathogenic impact of the SZ-risk DGCR2 mutation in mouse corticogenesis using in utero electroporation targeted to projection neurons. RESULTS: Dgcr2 knockdown impaired radial locomotion and final translocation of projection neurons, leading to persistent laminar positioning alterations. The DGCR2 missense SZ-risk mutation had a pathogenic impact on projection neuron laminar allocation by reducing protein expression. Mechanistically, we identified Dgcr2 as a novel member of the Reelin complex, regulating the phosphorylation of Reelin-dependent substrates and the expression of Reelin-dependent transcriptional targets. CONCLUSIONS: Overall, this study provides biological evidence that the SZ-risk gene DGCR2 regulates critical steps of early corticogenesis possibly through a Reelin-dependent mechanism. Additionally, we found that the SZ-risk mutation in DGCR2 has a pathogenic impact on cortical formation by reducing protein expression level, suggesting a functional role for DGCR2 haploinsufficiency in the 22q11.2 deletion syndrome.

The VWF-GPIb axis in ischaemic stroke: lessons from animal models.

Stroke is a leading cause of death and long-term disability worldwide. Ischaemic stroke is caused by a blood clot that obstructs cerebral blood flow. Current treatment mainly consists of achieving fast reperfusion, either via pharmacological thrombolysis using tissue plasminogen activator or via endovascular thrombectomy. Unfortunately, reperfusion therapy is only available to a limited group of patients and reperfusion injury can further aggravate brain damage. Hence, there is an urgent need for better understanding of ischaemic stroke pathophysiology in order to develop novel therapeutic strategies. In recent years, the pathophysiological importance of von Willebrand factor (VWF) in ischaemic stroke has become clear from both clinical and experimental studies. In particular, binding of VWF to platelet glycoprotein Ib (GPIb) has become an interesting target for ischaemic stroke therapy. Recent insights show that inhibting the VWF-GPIb interaction could result in a pro-thrombolytic activity improving cerebral reperfusion rates and concurrently reducing cerebral ischaemia/reperfusion damage. This review gives an overview of the experimental evidence that illustrates the crucial role of the VWF-GPIb axis in ischaemic stroke.

Thrombin-induced reactive oxygen species generation in platelets: A novel role for protease-activated receptor 4 and GPIbα.

BACKGROUND: Platelets are essential for maintaining haemostasis and play a key role in the pathogenesis of cardiovascular disease. Upon ligation of platelet receptors through subendothelial matrix proteins, intracellular reactive oxygen species (ROS) are generated, further amplifying the platelet activation response. Thrombin, a potent platelet activator, can signal through GPIbα and protease-activated receptor (PAR) 1 and PAR4 on human platelets, and recently has been implicated in the generation of ROS. While ROS are known to have key roles in intra-platelet signalling and subsequent platelet activation, the precise receptors and signalling pathways involved in thrombin-induced ROS generation have yet to be fully elucidated. OBJECTIVE: To investigate the relative contribution of platelet GPIbα and PARs to thrombin-induced reactive oxygen species (ROS) generation. METHODS AND RESULTS: Highly specific antagonists targeting PAR1 and PAR4, and the GPIbα-cleaving enzyme, Naja kaouthia (Nk) protease, were used in quantitative flow cytometry assays of thrombin-induced ROS production. Antagonists of PAR4 but not PAR1, inhibited thrombin-derived ROS generation. Removal of the GPIbα ligand binding region attenuated PAR4-induced and completely inhibited thrombin-induced ROS formation. Similarly, PAR4 deficiency in mice abolished thrombin-induced ROS generation. Additionally, GPIbα and PAR4-dependent ROS formation were shown to be mediated through focal adhesion kinase (FAK) and NADPH oxidase 1 (NOX1) proteins. CONCLUSIONS: Both GPIbα and PAR4 are required for thrombin-induced ROS formation, suggesting a novel functional cooperation between GPIbα and PAR4. Our study identifies a novel role for PAR4 in mediating thrombin-induced ROS production that was not shared by PAR1. This suggests an independent signalling pathway in platelet activation that may be targeted therapeutically.

Imaging the dynamic platelet-neutrophil response in sterile liver injury and repair in mice.

UNLABELLED: Although platelets have been extensively studied in hemostasis and inflammation, their role is not well understood in sterile liver injury and repair. Using a thermally induced focal liver injury and repair model and multichannel spinning disk confocal microscopy allowed visualization of the dynamic behavior of platelets and neutrophils in this insult. Platelets instantaneously adhered to molecularly altered sinusoidal endothelium adjacent to the afflicted area, paving approximately 200 µm abutting the injury. Platelets remained adherent for at least 4 hours, but dissipated by 8 hours. The early recruitment occurred by GPIIbIIIa (CD41) and the later recruitment was dependent upon both GPIIbIIIa and GPIb (CD42B). Platelets did not occlude the vessels, but rather paved the altered endothelium. Endothelin-induced vasoconstriction by hepatic stellate cells, and not platelet accumulation or coagulation, was responsible for temporarily restricted perfusion around the injury. Neutrophils crawled into the injury from significant distances through the sinusoids. The crawling neutrophils required the platelet-paved endothelium given that very little neutrophil recruitment was noted in thrombocytopenic or CD41-deficient mice. As platelets slowly dissipated, neutrophil recruitment was also halted. Previous work suggested that platelets binding to immobilized neutrophils induced neutrophil extracellular trap (NET) formation in response to infection as well as during thrombosis and other forms of sterile injury. In this model of neutrophils crawling on immobilized platelets, very few NETs were observed and no additional injury was noted. In fact, GPIIbIIIa-deficient mice had delayed repair. CONCLUSION: In a liver model of sterile injury and repair, platelets play a critical role in forming a substratum and pave the way for neutrophils to enter the injured site for subsequent repair.

Structure and function of platelet receptors initiating blood clotting.

At the clinical level, recent studies reveal the link between coagulation and other pathophysiological processes, including platelet activation, inflammation, cancer, the immune response, and/or infectious diseases. These links are likely to underpin the coagulopathy associated with risk factors for venous thromboembolic (VTE) and deep vein thrombosis (DVT). At the molecular level, the interactions between platelet-specific receptors and coagulation factors could help explain coagulopathy associated with aberrant platelet function, as well as revealing new approaches targeting platelet receptors in diagnosis or treatment of VTE or DVT. Glycoprotein (GP)Ibα, the major ligand-binding subunit of the platelet GPIb-IX-V complex, that binds the adhesive ligand, von Willebrand factor (VWF), is co-associated with the platelet-specific collagen receptor, GPVI. The GPIb-IX-V/GPVI adheso-signaling complex not only initiates platelet activation and aggregation (thrombus formation) in response to vascular injury or disease but GPIbα also regulates coagulation through a specific interaction with thrombin and other coagulation factors. Here, we discuss the structure and function of key platelet receptors involved in thrombus formation and coagulation in health and disease, with a particular focus on platelet GPIbα.

Streptococcus pneumoniae worsens cerebral ischemia via interleukin 1 and platelet glycoprotein Ibα.

OBJECTIVE: Bacterial infection contributes to diverse noninfectious diseases and worsens outcome after stroke. Streptococcus pneumoniae, the most common infection in patients at risk of stroke, is a major cause of prolonged hospitalization and death of stroke patients, but how infection impacts clinical outcome is not known. METHODS: We induced sustained pulmonary infection by a human S. pneumoniae isolate in naive and comorbid rodents to investigate the effect of infection on vascular and inflammatory responses prior to and after cerebral ischemia. RESULTS: S. pneumoniae infection triggered atherogenesis, led to systemic induction of interleukin (IL) 1, and profoundly exacerbated (50-90%) ischemic brain injury in rats and mice, a response that was more severe in combination with old age and atherosclerosis. Systemic blockade of IL-1 with IL-1 receptor antagonist (IL-1Ra) fully reversed infection-induced exacerbation of brain injury and functional impairment caused by cerebral ischemia. We show that infection-induced systemic inflammation mediates its effects via increasing platelet activation and microvascular coagulation in the brain after cerebral ischemia, as confirmed by reduced brain injury in response to blockade of platelet glycoprotein (GP) Ibα. IL-1 and platelet-mediated signals converge on microglia, as both IL-1Ra and GPIbα blockade reversed the production of IL-1α by microglia in response to cerebral ischemia in infected animals. INTERPRETATION: S. pneumoniae infection augments atherosclerosis and exacerbates ischemic brain injury via IL-1 and platelet-mediated systemic inflammation. These mechanisms may contribute to diverse cardio- and cerebrovascular pathologies in humans.

Specific inhibition of ectodomain shedding of glycoprotein Ibα by targeting its juxtamembrane shedding cleavage site.

BACKGROUND: Ectodomain shedding of glycoprotein Ibα (GPIbα), a proteolytic event in which metalloprotease ADAM17 cleaves the Gly464-Val465 bond and releases glycocalicin to the plasma, is considered a critical step in mediating clearance of stored platelets. Supporting evidence has largely come from studies using ADAM17 inhibitors. However, the definitive proof is lacking due to the broad substrate specificity of ADAM17. AIM: To achieve substrate-specific inhibition of GPIbα shedding. METHODS: Development of monoclonal antibodies that directly bind the sequence around the GPIbα shedding cleavage site and inhibit GPIbα shedding by blocking ADAM17 access to the cleavage site. RESULTS: Six anti-GPIbα monoclonal antibodies with varying binding affinities were obtained. The prototypic clone, designated 5G6, and its monomeric Fab fragment bind specifically purified GPIb-IX complex, human platelets, and transgenic murine platelets expressing human GPIbα. The clone 5G6 showed similar inhibitory potency as a widely used shedding inhibitor GM6001 in both constitutive and induced GPIbα shedding in human platelets. It does not recognize mouse GPIbα or inhibit shedding of other platelet receptors. Finally, 5G6 binding displays no detectable effect on platelet activation and aggregation. CONCLUSIONS: The clone 5G6 specifically inhibits GPIbα shedding with no detectable effect on platelet functions. The method of substrate-specific shedding inhibition by macromolecular binding of the shedding cleavage site can be applicable to many other transmembrane receptors undergoing ectodomain shedding.

Dietary α-linolenic acid increases the platelet count in ApoE-/- mice by reducing clearance.

Previously we reported that dietary intake of alpha-linolenic acid (ALA) reduces atherogenesis and inhibits arterial thrombosis. Here, we analyze the substantial increase in platelet count induced by ALA and the mechanisms of reduced platelet clearance. Eight-week-old male apolipoprotein E knockout (ApoE(-/-)) mice were fed a 0.21g% cholesterol diet complemented by either a high- (7.3g%) or low-ALA (0.03g%) content. Platelet counts doubled after 16 weeks of ALA feeding, whereas the bleeding time remained similar. Plasma glycocalicin and glycocalicin index were reduced, while reticulated platelets, thrombopoietin, and bone marrow megakaryocyte colony-forming units remained unchanged. Platelet contents of liver and spleen were substantially reduced, without affecting macrophage function and number. Glycoprotein Ib (GPIb) shedding, exposure of P-selectin, and activated integrin αIIbß3 upon activation with thrombin were reduced. Dietary ALA increased the platelet count by reducing platelet clearance in the reticulo-endothelial system. The latter appears to be mediated by reduced cleavage of GPIb by tumor necrosis factor-α-converting enzyme and reduced platelet activation/expression of procoagulant signaling. Ex vivo, there was less adhesion of human platelets to von Willebrand factor under high shear conditions after ALA treatment. Thus, ALA may be a promising tool in transfusion medicine and in high turnover/high activation platelet disorders.

Glycoprotein Ibα and FcγRIIa play key roles in platelet activation by the colonizing bacterium, Streptococcus oralis.

BACKGROUND: Infective endocarditis (IE) is characterized by thrombus formation on a cardiac valve. The oral bacterium, Streptococcus oralis, is recognized for its ability to colonize damaged heart valves and is frequently isolated from patients with IE. Platelet interaction with S. oralis leads to the development of a thrombotic vegetation on heart valves, which results in valvular incompetence and congestive heart failure. OBJECTIVE: To investigate the mechanism through which platelets become activated upon binding S. oralis. PATIENTS AND METHODS: Platelet interactions with immobilized bacteria under shear conditions were assessed using a parallel flow chamber. S. oralis-inducible platelet reactivity was determined using light transmission aggregometry. Dense granule secretion was measured by luminometry using a luciferin/luciferase assay. RESULTS: Using shear rates that mimic physiological conditions, we demonstrated that S. oralis was able to support platelet adhesion under venous (50-200 s(-1) ) and arterial shear conditions (800 s(-1) ). Platelets rolled along immobilized S. oralis through an interaction with GPIbα. Following rolling, platelet microaggregate formation was observed on immobilized S. oralis. Aggregate formation was dependent on S. oralis binding IgG, which cross-links to platelet FcγRIIa. This interaction led to phosphorylation of the ITAM domain on FcγRIIa, resulting in dense granule secretion, amplification through the ADP receptor and activation of RAP1, culminating in platelet microaggregate formation. CONCLUSIONS: These results suggest a model of interaction between S. oralis and platelets that leads to the formation of a stable septic vegetation on damaged heart valves.

The organizing principle of the platelet glycoprotein Ib-IX-V complex.

The glycoprotein (GP)Ib-IX-V complex is the platelet receptor for von Willebrand factor and many other molecules that are critically involved in hemostasis and thrombosis. The lack of functional GPIb-IX-V complexes on the platelet surface is the cause of Bernard-Soulier syndrome, a rare hereditary bleeding disorder that is also associated with macrothrombocytopenia. GPIb-IX-V contains GPIbα, GPIbß, GPIX and GPV subunits, all of which are type I transmembrane proteins containing leucine-rich repeat domains. Although all of the subunits were identified decades ago, not until recently did the mechanism of complex assembly begin to emerge from a systematic characterization of inter-subunit interactions. This review summarizes the forces driving the assembly of GPIb-IX-V, discusses their implications for the pathogenesis of Bernard-Soulier syndrome, and identifies questions that remain about the structure and organization of GPIb-IX-V.

Platelets with a W127X mutation in GPIX express sufficient residual amounts of GPIbα to support adhesion to von Willebrand factor and collagen.

Bernard-Soulier syndrome (BSS) is an inherited bleeding disorder caused by a defect in the platelet glycoprotein (GP) Ib/IX complex. The GPIX W127X mutation is the most common genetic defect in Japanese patients with BSS, which is often misdiagnosed as immune thrombocytopenic purpura, presumably due to residual expression of GPIbα. Neither the mechanism by which this mutation leads to a mild bleeding diathesis, nor whether functional GPIbα is expressed on platelet surfaces is known. We investigated GPIbα expression and function in platelets with a GPIX W127X mutation (GPIXW127X). GPIbα complexed with GPIbß by disulfide bonding was expressed on GPIXW127X platelets and stable CHO-K1 cells lacking GPIX but expressing GPIbα and GPIbß. Expression of GPIbα/ß on GPIXW127X platelets was sufficient to support adhesion to immobilized von Willebrand factor and type III collagen and ristocetin-induced platelet agglutination. A residual amount of functional GPIbα/ß heteromer expressed on GPIXW127X platelets partially compensates for the absence of the GPIb/IX complex. This may account for the mild bleeding phenotype of the BSS variant characterized by a non-sense mutation in GPIX.

Inhibitors of the interaction between von Willebrand factor and platelet GPIb/IX/V.

The formation of platelet-rich thrombi, a critical step in the pathogenesis of atherothrombotic events, is a multistep process involving several components, among which von Willebrand Factor (VWF) plays a central role. Ruptured atherosclerotic plaques expose subendothelial matrix proteins which bind VWF that represents a bridge between the injured blood vessel and activated platelets, playing a crucial role in platelet adhesion and aggregation, especially in conditions of high-shear rate. Due to these peculiarities, the binding of VWF to GPIbα is an attractive drug target. Here we summarize the present knowledge on the different classes of drugs targeting the VWF-GPIb interaction and we give an account of their level of clinical development. In particular, the following compounds are discussed: AJW200, an IgG4 humanized monoclonal antibody against VWF-A1; 82D6A3, a monoclonal antibody against VWF-A3; ALX-0081 and ALX-0681, bivalent humanized nanobodies targeting the VWF-A1 domain; ARC1779 and its advanced formulation ARC15105, second-generation aptamers that bind the VWF-A1 domain; h6B4-Fab, a murine monoclonal antibody, and GPG-290, a recombinant chimeric protein, both directed against GPIbα.

The CX3C chemokine fractalkine mediates platelet adhesion via the von Willebrand receptor glycoprotein Ib.

The membrane-anchored CX3C chemokine fractalkine (FKN) is expressed on activated endothelium and is associated with the development of atherosclerosis. The potential of FKN in mediating platelet adhesion beyond platelet activation remains unexplored to date. A flow-based adhesion assay was used to study the adhesion of platelets to immobilized FKN under physiologic flow conditions. Platelet adhesion to von Willebrand factor (VWF) was increased in the presence of FKN at 600 inverse seconds. Additional platelet adhesion to FKN coimmobilized with VWF was dependent on the FKN receptor CX3CR1 and activation of glycoprotein (GP) IIb/IIIa. The number of platelets rolling on VWF was likewise enhanced in the presence of FKN. The enhancement of rolling on FKN and VWF was insensitive to anti-CX3CR1 antibody but was fully inhibited by neutralizing GPIbα function. The extracellular domain of GPIbα was covalently coupled to fluorescent microspheres, and microsphere binding was significantly higher in the presence of FKN. Platelet adhesion to activated endothelium in vitro and to intact human arteries was substantially increased in an FKN-dependent manner. These data demonstrate that endothelial expressed FKN activates platelets via its cognate receptor CX3CR1, whereas platelet adhesion is predominantly mediated by GPIbα and independent of CX3CR1.

Platelet adhesion to collagen.

Platelets play a central role in maintaining hemostasis mainly by binding to subendothelial collagen exposed upon vascular injury, thereby initiating thrombus formation. Platelets can bind directly to the exposed collagen through two major receptors i.e. the integrin a2b1 and glycoprotein (GP) VI. However, under high shear conditions the GPIb-V-IX receptor complex and its main ligand von Willebrand Factor are additionally needed for firm platelet adhesion to the vessel wall. In this review, we summarize the current knowledge on the individual roles and structure-function relationships of these main platelet adhesion receptors.

Targeting von Willebrand factor and platelet glycoprotein Ib receptor.

Atherothrombotic events, such as acute coronary syndrome or stroke, are the result of platelet activation. Von Willebrand factor (vWF), a multimeric glycoprotein, plays a key role in aggregation of platelets, especially under high-shear conditions. Acting as bridging element or ligand between damaged endothelial sites and the glycoprotein Ib (GPIb) receptor on platelets, vWF is responsible for platelet adhesion and aggregation. This vWF activation and further platelet aggregation mainly occurs under high shear stress present in small arterioles or during deficiency of the vWF-cleaving protease ADAMTS13. There are several substances targeting vWF itself or its binding receptor GPIb on platelets. Two antibodies are directed against vWF: AJW200, an IgG4 humanized monoclonal antibody, and 82D6A3, a monoclonal antibody of the collagen-binding A-3 domain of vWF. ALX-0081 and ALX-0681 are bivalent humanized nanobodies targeting the GPIb binding site of vWF. Aptamers are oligonucleotides with drug-like properties that share some of the attributes of monoclonal antibodies. ARC1779 is a second-generation, nuclease-resistant aptamer, binding to the activated vWF A1 domain and ARC15105 is a chemically advanced follower with an assumed higher affinity to vWF. Antibodies targeting GPIbα are h6B4-Fab, a murine monoclonal antibody; GPG-290, a recombinant, chimeric protein containing the amino-terminal 290 amino acids of GPIbα linked to human IgG1 Fc; and the monoclonal antibody SZ2. There are a number of promising preclinical results and development of some agents (AJW 200, ARC1779 and ALX-0081) has already reached Phase II trials.

The role of glycoprotein Ibalpha and von Willebrand factor interaction in stroke development.

Ischaemic stroke is a devastating disease with limited treatment options due to numerous uncertainties regarding the underlying pathophysiology. The contribution of glycoprotein (GP)Ibalpha and von Willebrand factor (VWF) in stroke development has only recently been established in mice. Complete blockade of GPIbalpha led to a significant reduction of infarct volumes in mice undergoing one hour of transient middle cerebral artery occlusion (tMCAO). High shear-induced changes in VWF confirmation are a prerequisite for VWF binding to collagen and GPIbalpha expressed on platelets. Importantly, transgenic VWF-/- mice were similarly protected against ischemic stroke after tMCAO, and hydrodynamic injection of a VWF-encoding plasmid restored VWF serum levels and the susceptibility towards stroke. Secreted VWF is rapidly cleaved by ADAMTS13. Accordingly, ADAMTS13 deficient mice developed larger infarction after tMCAO, while infusion of recombinant ADAMTS13 into wild-type mice was stroke-protective. In conclusion, there is compelling evidence that GPIbalpha/VWF interactions and downstream signaling via phospholipase D1 (PLD1) provide new therapeutic targets in ischemic stroke.

A central role of GPIb-IX in the procoagulant function of platelets that is independent of the 45-kDa GPIbalpha N-terminal extracellular domain.

Activated platelets become procoagulant and efficiently promote the conversion of prothrombin to thrombin. A role of the GPIb-V-IX complex has long been postulated in view of the decreased prothrombin consumption in Bernard-Soulier patients. We evaluated the impact of GPIb-V-IX deficiency and the requirement for the GPIbalpha extracellular domain. In GPIbbeta(-/-) mice, thrombin generation was profoundly decreased in tissue factor- or collagen-related peptide (CRP)-activated platelet-rich plasma and in washed platelets supplemented with normal plasma or with FVa, FXa, and prothrombin. Phosphatidylserine (PS) exposure was similarly decreased in response to thrombin, CRP, or CRP + PAR4 peptide despite a normal platelet phospholipid composition. The hypothesis that these defects originate from lack of the GPIbalpha N-terminal domain was evaluated after its removal from normal mouse and human platelets with Nk protease or O-sialoglycoprotein endopeptidase. Unexpectedly, the treated platelets exhibited normal thrombin generation and PS exposure, indicating that GPIb-V-IX regulates procoagulant activity independently of its GPIbalpha-binding region. These results suggested a more general structuring role through intracellular cytoskeleton-anchoring portions regulating responses leading to PS exposure. This hypothesis was supported by the decreased calcium mobilization observed in GPIbbeta(-/-) platelets in response to several agonists, some acting independently of GPIb, in contrast to the normal calcium responses in Nk protease-treated platelets.