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Background: Thrombocytopenia has been consistently described in patients with extracorporeal membrane oxygenation (ECMO) and associated with poor outcome. However, the prevalence and underlying mechanisms remain largely unknown, and a device-related role of ECMO in thrombocytopenia has been hypothesized. This study aims to investigate the mechanisms underlying thrombocytopenia in ECMO patients., Methods: In a prospective cohort of 107 ECMO patients, we investigated platelet count, functions, and glycoprotein shedding. In an ex vivo mock circulatory ECMO loop, we assessed platelet responses and VWF (von Willebrand factor)-GP Ibα (glycoprotein Ibα) interactions at low- and high-flow rates, in the presence or absence of red blood cells. The clearance of human platelets subjected or not to ex vivo perfusion was studied using an in vivo transfusion model in NOD/SCID (nonobese diabetic/severe combined Immunodeficient) mice., Results: In ECMO patients, we observed a time-dependent decrease in platelet count starting 1 hour after device onset, with a mean drop of 7%, 35%, and 41% at 1, 24, and 48 hours post-ECMO initiation ( P =0.00013, P <0.0001, and P <0.0001, respectively), regardless of the type of ECMO. This drop in platelet count was associated with a decrease in platelet GP Ibα expression (before: 47.8±9.1 versus 24 hours post-ECMO: 42.3±8.9 mean fluorescence intensity; P =0.002) and an increase in soluble GP Ibα plasma levels (before: 5.6±3.3 versus 24 hours post-ECMO: 10.8±4.1 µg/mL; P <0.0001). GP Ibα shedding was also observed ex vivo and was unaffected by (1) red blood cells, (2) the coagulation potential, (3) an antibody blocking VWF-GP Ibα interaction, (4) an antibody limiting VWF degradation, and (5) supraphysiological VWF plasma concentrations. In contrast, GP Ibα shedding was dependent on rheological conditions, with a 2.8-fold increase at high- versus low-flow rates. Platelets perfused at high-flow rates before being transfused to immunodeficient mice were eliminated faster in vivo with an accelerated clearance of GP Ibα-negative versus GP Ibα-positive platelets., Conclusions: ECMO-associated shear forces induce GP Ibα shedding and thrombocytopenia due to faster clearance of GP Ibα-negative platelets. Inhibiting GP Ibα shedding could represent an approach to reduce thrombocytopenia during ECMO., Competing Interests: Disclosures None.

Rationale: Ca 2+ signaling is a key and ubiquitous actor of cell organization and its modulation controls many cellular responses. SERCAs (sarco-endoplasmic reticulum Ca 2+ -ATPases) pump Ca 2+ into internal stores that play a major role in the cytosolic Ca 2+ concentration rise upon cell activation. Platelets exhibit 2 types of SERCAs, SERCA2b and SERCA3 (SERCA3 deficient mice), which may exert specific roles, yet ill-defined. We have recently shown that Ca 2+ mobilization from SERCA3-dependent stores was required for full platelet activation in weak stimulation conditions., Objective: To uncover the signaling mechanisms associated with Ca 2+ mobilization from SERCA3-dependent stores leading to ADP secretion., Methods and Results: Using platelets from wild-type or Serca3 -deficient mice, we demonstrated that an early (within 5-10 s following stimulation) secretion of ADP specifically dependent on SERCA3 stored Ca 2+ is exclusively mobilized by nicotinic acid adenosine dinucleotide-phosphate (NAADP): both Ca 2+ mobilization from SERCA3-dependent stores and primary ADP secretion are blocked by the NAADP receptor antagonist Ned-19, and reciprocally both are stimulated by permeant NAADP. In contrast, Ca 2+ mobilization from SERCA3-dependent stores and primary ADP secretion were unaffected by inhibition of the production of IP3 (inositol-1,4,5-trisphosphate) by phospholipase-C and accordingly were not stimulated by permeant IP3., Conclusions: Upon activation, an NAADP/SERCA3 Ca 2+ mobilization pathway initiates an early ADP secretion, potentiating platelet activation, and a secondary wave of ADP secretion driven by both an IP3/SERCA2b-dependent Ca 2+ stores pathway and the NAADP/SERCA3 pathway. This does not exclude that Ca 2+ mobilized from SERCA3 stores may also enhance platelet global reactivity to agonists. Because of its modulating effect on platelet activation, this NAADP-SERCA3 pathway may be a relevant target for anti-thrombotic therapy. Graphic Abstract: A graphic abstract is available for this article.

Background: Platelet cross-linking during arterial thrombosis involves von Willebrand Factor (VWF) multimers. Therefore, proteolysis of VWF appears promising to disaggregate platelet-rich thrombi and restore vessel patency in acute thrombotic disorders such as ischemic stroke, acute coronary syndrome, or acute limb ischemia. N -Acetylcysteine (NAC, a clinically approved mucolytic drug) can reduce intrachain disulfide bonds in large polymeric proteins. In the present study, we postulated that NAC might cleave the VWF multimers inside occlusive thrombi, thereby leading to their dissolution and arterial recanalization., Methods: Experimental models of thrombotic stroke induced by either intra-arterial thrombin injection or ferric chloride application followed by measurement of cerebral blood flow using a combination of laser Doppler flowmetry and MRI were performed to uncover the effects of NAC on arterial thrombi. To investigate the effect of NAC on larger vessels, we also performed ferric chloride-induced carotid artery thrombosis. In vitro experiments were performed to study the molecular bases of NAC thrombolytic effect, including platelet aggregometry, platelet-rich thrombi lysis assays, thromboelastography (ROTEM), and high-shear VWF string formation using microfluidic devices. We also investigated the putative prohemorrhagic effect of NAC in a mouse model of intracranial hemorrhage induced by in situ collagenase type VII injection., Results: We demonstrated that intravenous NAC administration promotes lysis of arterial thrombi that are resistant to conventional approaches such as recombinant tissue-type plasminogen activator, direct thrombin inhibitors, and antiplatelet treatments. Through in vitro and in vivo experiments, we provide evidence that the molecular target underlying the thrombolytic effects of NAC is principally the VWF that cross-link platelets in arterial thrombi. Coadministration of NAC and a nonpeptidic GpIIb/IIIa inhibitor further improved its thrombolytic efficacy, essentially by accelerating thrombus dissolution and preventing rethrombosis. Thus, in a new large-vessel thromboembolic stroke model in mice, this cotreatment significantly improved ischemic lesion size and neurological outcome. It is important to note that NAC did not worsen hemorrhagic stroke outcome, suggesting that it exerts thrombolytic effects without significantly impairing normal hemostasis., Conclusions: We provide evidence that NAC is an effective and safe alternative to currently available antithrombotic agents to restore vessel patency after arterial occlusion., (© 2017 American Heart Association, Inc.)

Background: Laminins are major components of basement membranes, well located to interact with platelets upon vascular injury. Laminin-111 (α1β1γ1) is known to support platelet adhesion but is absent from most blood vessels, which contain isoforms with the α2, α4, or α5 chain. Whether vascular laminins support platelet adhesion and activation and the significance of these interactions in hemostasis and thrombosis remain unknown., Methods and Results: Using an in vitro flow assay, we show that laminin-411 (α4β1γ1), laminin-511 (α5β1γ1), and laminin-521 (α5β2γ1), but not laminin-211 (α2β1γ1), allow efficient platelet adhesion and activation across a wide range of arterial wall shear rates. Adhesion was critically dependent on integrin α6β1 and the glycoprotein Ib-IX complex, which binds to plasmatic von Willebrand factor adsorbed on laminins. Glycoprotein VI did not participate in the adhesive process but mediated platelet activation induced by α5-containing laminins. To address the significance of platelet/laminin interactions in vivo, we developed a platelet-specific knockout of integrin α6. Platelets from these mice failed to adhere to laminin-411, laminin-511, and laminin-521 but responded normally to a series of agonists. α6β1-Deficient mice presented a marked decrease in arterial thrombosis in 3 models of injury of the carotid, aorta, and mesenteric arterioles. The tail bleeding time and blood loss remained unaltered, indicating normal hemostasis., Conclusions: This study reveals an unsuspected important contribution of laminins to thrombus formation in vivo and suggests that targeting their main receptor, integrin α6β1, could represent an alternative antithrombotic strategy with a potentially low bleeding risk.

Objective: To unravel crucial von Willebrand factor (VWF) interactions that are detrimental in stroke development., Methods and Results: VWF(-/-) mice received gene transfer to express mutants of VWF defective either in binding to fibrillar collagen, glycoprotein (GP)Ibα or GPIIb/IIIa, and underwent 60 minutes of transient middle cerebral artery occlusion. In VWF(-/-) mice reconstituted with VWF mutants defective in binding to collagen or GPIbα, protection against stroke was sustained, whereas VWF lacking the GPIIb/IIIa binding site restored full susceptibility similar to normal VWF., Conclusions: VWF-collagen and VWF-GPIbα (but not VWF-GPIIb/IIIa) interactions are instrumental in thrombus formation after transient middle cerebral artery occlusion, and their inhibition could be a promising target for stroke treatment.

Objective: von Willebrand Factor (vWF) structure-function relationship has been studied only in vitro. To investigate the physiological importance of particular vWF domains, we have introduced mutations into murine vWF (mvWF) cDNA inhibiting vWF binding to glycoprotein (Gp) Ib, GpIIbIIIa, and to fibrillar collagen., Methods and Results: We delivered wild-type (WT) or mutant mvWF cDNA into vWF-deficient (Vwf-/-) mice using hydrodynamic injection and assessed whether hemorrhagic symptoms could be corrected. Hydrodynamic gene transfer resulted in high expression of plasma mvWF 24 hours after injection (438+/-63% for 50 microg of cDNA). Factor VIII activity was normalized in Vwf-/- mice injected with mvWF cDNA and multimerization was achieved. Bleeding time was corrected after injection of WT mvWF cDNA in Vwf-/- mice whereas noninjected mice did not stop bleeding. Injection of the GpIIbIIIa and the collagen binding mutants in Vwf-/- mice also resulted in a correction of bleeding time whereas mice injected with the GpIb binding mutant were bleeding for as long they were observed, although blood loss was decreased compared with noninjected mice (61+/-21 microL versus 232+/-63 microL)., Conclusions: Our model allows the rapid in vivo evaluation of specific mutations on plasma vWF function.

Platelet adhesion and aggregation at sites of vascular injury are two key events in hemostasis and thrombosis. Because of exciting advances in genetic engineering, the mouse has become an important and frequently used model to unravel the molecular mechanisms underlying the multistep process leading to the formation of a stable platelet plug. In gene-targeted mice, the crucial importance of platelet adhesion receptors such as glycoprotein Ib alpha or the alphaIIb beta3 integrin has been confirmed and further clarified. Their absence leads to highly impaired thrombus formation, independent of the model used to induce vascular injury. In contrast, the relative contribution of other receptors, such as glycoprotein VI, or of various platelet ligands may be regulated by the severity of injury, the type of vessel injured, and the signaling pathways that are generated. Murine models have also helped improve understanding of the second wave of events that leads to stabilization of the platelet aggregate. Despite the current limitations due to lack of standardization and the virtual absence of thrombosis models in diseased vessels, there is no doubt that the mouse will play a key role in the discovery and characterization of the next generation of antithrombotic agents. This review focuses on key findings about the molecular mechanisms supporting hemostasis and thrombosis that have been obtained with genetically engineered mouse models deficient in various platelet adhesion receptors and ligands. Combination of these models with sophisticated methods allowing direct visualization of platelet-vessel wall interactions after injury greatly contributed to recent advances in the field.

Objective: To explore the effect of the Heerlen polymorphism and C4b-binding protein (C4BP) on protein S catabolism in vitro and in vivo., Methods and Results: Radiolabeled protein S was efficiently bound and intracellularly degraded by THP-1 macrophages, and both processes were strongly reduced in the presence of the protein S-carrier protein C4BP. To test whether C4BP displays a similar protective effect in vivo, survival experiments were performed in mice. In the absence of C4BP, radiolabeled human protein S disappeared in a biphasic manner (mean residence time [MRT] 2 hours). However, the presence of C4BP resulted in a 4-fold prolonged survival of protein S (MRT 8 hours; P<0.0001). We also applied this experimental model to recombinant protein S-Heerlen, a naturally occurring variant that contains a Ser460Pro substitution. These clearance experiments revealed a strongly decreased survival of recombinant protein S-S460P (MRT 0.6 hours; P=0.021), which could be compensated partially by C4BP (MRT 1.4 hours; P=0.012 compared with protein S-S460P)., Conclusions: Protein S-S460P has a reduced survival in vivo, which may explain the low levels of free protein S in individuals carrying this polymorphism. Furthermore, C4BP prevents premature clearance of protein S and uses this ability to compensate the increased clearance of protein S-S460P.