[Platelet adhesion mediated by von Willebrand factor in patients with premature coronary artery disease].

Aim    To study platelet adhesion mediated by von Willebrand factor (VWF) in patients with premature ischemic heart disease (IHD).Material and methods    This study enrolled 58 patients with stable IHD, including 45 men younger than 55 years with the first manifestation of IHD at the age of <50 years and 13 women younger than 65 years with the first manifestation of IHD at the age of <60 years. The control group consisted of 33 patients, 13 men younger than 55 years and 20 women younger than 65 years without IHD. Platelet adhesion to the collagen surface at the shear rate of 1300 s-1 was studied by evaluating the intensity of scattered laser light from the collagen-coated optical substrate in a flow chamber of a microfluidic device after 15-min circulation of whole blood in the chamber. Decreases in platelet adhesion after addition to the blood of monoclonal antibodies (mAb) to platelet receptors glycoproteins Ib (GPIb) to inhibit the receptor interaction with VWF were compared for patients of both groups. Results    In patients with premature IHD, the decrease in platelet adhesion following the platelet GPIb receptor inhibition was significantly less than in patients of the control group (74.8 % (55.6; 82.7) vs. 28.9 % (-9.8; 50,5), p <0.001). For the entire sample, the median decrease in platelet adhesion following the GPIb receptor inhibition was 62.8 % (52.2; 71.2). With an adjustment for traditional risk factors of IHD, a decrease in platelet adhesion of >62.8% after blocking GPIb receptors increased the likelihood of premature IHD (OR=9.84, 95 % CI: 2.80-34.59; p <0.001).Conclusion    Blocking the interaction of GPIb receptors with VWF in patients with premature IHD and increased shear rate induced a greater decrease in platelet adhesion than in patients without this disease. This suggested that an excessive interaction of VWF with platelets might contribute to the pathogenesis of premature IHD.

Structure modeling hints at a granular organization of the Golgi ribbon.

BACKGROUND: In vertebrate cells, the Golgi functional subunits, mini-stacks, are linked into a tri-dimensional network. How this "ribbon" architecture relates to Golgi functions remains unclear. Are all connections between mini-stacks equal? Is the local structure of the ribbon of functional importance? These are difficult questions to address, without a quantifiable readout of the output of ribbon-embedded mini-stacks. Endothelial cells produce secretory granules, the Weibel-Palade bodies (WPB), whose von Willebrand Factor (VWF) cargo is central to hemostasis. The Golgi apparatus controls WPB size at both mini-stack and ribbon levels. Mini-stack dimensions delimit the size of VWF "boluses" whilst the ribbon architecture allows their linear co-packaging, thereby generating WPBs of different lengths. This Golgi/WPB size relationship suits mathematical analysis. RESULTS: WPB lengths were quantized as multiples of the bolus size and mathematical modeling simulated the effects of different Golgi ribbon organizations on WPB size, to be compared with the ground truth of experimental data. An initial simple model, with the Golgi as a single long ribbon composed of linearly interlinked mini-stacks, was refined to a collection of mini-ribbons and then to a mixture of mini-stack dimers plus long ribbon segments. Complementing these models with cell culture experiments led to novel findings. Firstly, one-bolus sized WPBs are secreted faster than larger secretory granules. Secondly, microtubule depolymerization unlinks the Golgi into equal proportions of mini-stack monomers and dimers. Kinetics of binding/unbinding of mini-stack monomers underpinning the presence of stable dimers was then simulated. Assuming that stable mini-stack dimers and monomers persist within the ribbon resulted in a final model that predicts a "breathing" arrangement of the Golgi, where monomer and dimer mini-stacks within longer structures undergo continuous linking/unlinking, consistent with experimentally observed WPB size distributions. CONCLUSIONS: Hypothetical Golgi organizations were validated against a quantifiable secretory output. The best-fitting Golgi model, accounting for stable mini-stack dimers, is consistent with a highly dynamic ribbon structure, capable of rapid rearrangement. Our modeling exercise therefore predicts that at the fine-grained level the Golgi ribbon is more complex than generally thought. Future experiments will confirm whether such a ribbon organization is endothelial-specific or a general feature of vertebrate cells.

Arf GTPase-activating proteins SMAP1 and AGFG2 regulate the size of Weibel-Palade bodies and exocytosis of von Willebrand factor.

Arf GTPase-Activating proteins (ArfGAPs) mediate the hydrolysis of GTP bound to ADP-ribosylation factors (Arfs), which are critical to form transport intermediates. ArfGAPs have been thought to be negative regulators of Arfs; however, accumulating evidence indicates that ArfGAPs are important for cargo sorting and promote membrane traffic. Weibel-Palade bodies (WPBs) are cigar-shaped secretory granules in endothelial cells that contain von Willebrand factor (vWF) as their main cargo. WPB biogenesis at the Golgi was reported to be regulated by Arf and their regulators, but the role of ArfGAPs has been unknown. In this study, we performed siRNA screening of ArfGAPs to investigate the role of ArfGAPs in the biogenesis of WPBs. We found two ArfGAPs, SMAP1 and AGFG2, to be involved in WPB size and vWF exocytosis, respectively. SMAP1 depletion resulted in small-sized WPBs, and the lysosomal inhibitor leupeptin recovered the size of WPBs. The results indicate that SMAP1 functions in preventing the degradation of cigar-shaped WPBs. On the other hand, AGFG2 downregulation resulted in the inhibition of vWF secretion upon Phorbol 12-myristate 13-acetate (PMA) or histamine stimulation, suggesting that AGFG2 plays a role in vWF exocytosis. Our study revealed unexpected roles of ArfGAPs in vWF transport.

Conformation of the von Willebrand factor/factor VIII complex in quasi-static flow.

Von Willebrand factor (VWF) is a plasma glycoprotein that circulates noncovalently bound to blood coagulation factor VIII (fVIII). VWF is a population of multimers composed of a variable number of ∼280 kDa monomers that is activated in shear flow to bind collagen and platelet glycoprotein Ibα. Electron microscopy, atomic force microscopy, small-angle neutron scattering, and theoretical studies have produced a model in which the conformation of VWF under static conditions is a compact, globular "ball-of-yarn," implying strong, attractive forces between monomers. We performed sedimentation velocity (SV) analytical ultracentrifugation measurements on unfractionated VWF/fVIII complexes. There was a 20% per mg/ml decrease in the weight-average sedimentation coefficient, sw, in contrast to the ∼1% per mg/ml decrease observed for compact globular proteins. SV and dynamic light scattering measurements were performed on VWF/fVIII complexes fractionated by size-exclusion chromatography to obtain sw values and z-average diffusion coefficients, Dz. Molecular weights estimated using these values in the Svedberg equation ranged from 1.7 to 4.1 MDa. Frictional ratios calculated from Dz and molecular weights ranged from 2.9 to 3.4, in contrast to values of 1.1-1.3 observed for globular proteins. The Mark-Houwink-Kuhn-Sakurada scaling relationships between sw, Dz and molecular weight, [Formula: see text] and [Formula: see text] , yielded estimates of 0.51 and -0.49 for as and aD, respectively, consistent with a random coil, in contrast to the as value of 0.65 observed for globular proteins. These results indicate that interactions between monomers are weak or nonexistent and that activation of VWF is intramonomeric.

Conformation-dependent blockage of activated VWF improves outcomes of traumatic brain injury in mice.

Traumatic brain injury-induced coagulopathy (TBI-IC) causes life-threatening secondary intracranial bleeding. Its pathogenesis differs mechanistically from that of coagulopathy arising from extracranial injuries and hemorrhagic shock, but it remains poorly understood. We report results of a study designed to test the hypothesis that von Willebrand factor (VWF) released during acute TBI is intrinsically hyperadhesive because its platelet-binding A1-domain is exposed and contributes to TBI-induced vascular leakage and consumptive coagulopathy. This hyperadhesive VWF can be selectively blocked by a VWF A2-domain protein to prevent TBI-IC and to improve neurological function with a minimal risk of bleeding. We demonstrated that A2 given through intraperitoneal injection or IV infusion reduced TBI-induced death by >50% and significantly improved the neurological function of C57BL/6J male mice subjected to severe lateral fluid percussion injury. A2 protected the endothelium from extracellular vesicle-induced injury, reducing TBI-induced platelet activation and microvesiculation, and preventing a TBI-induced hypercoagulable state. A2 achieved this therapeutic efficacy by specifically blocking the A1 domain exposed on the hyperadhesive VWF released during acute TBI. These results suggest that VWF plays a causal role in the development of TBI-IC and is a therapeutic target for this life-threatening complication of TBI.

Role of Von Willebrand Factor after Injury: It May Do More Than We Think.

ABSTRACT: Acute traumatic coagulopathy is a complex phenomenon following injury and a main contributor to hemorrhage. It remains a leading cause of preventable death in trauma patients. This phenomenon is initiated by systemic injury to the vascular endothelium that is exacerbated by hypoperfusion, acidosis, and hypothermia and leads to systemic activation of the coagulation cascades and resultant coagulopathy. Many previous studies have focused on endotheliopathy with targeted markers such as syndecan-1, soluble thrombomodulin, and plasma adrenaline as potential culprits for initiation and propagation of this state. However, in more recent studies, hyperadhesive von Willebrand factor (VWF), which is released following endothelial injury, and its cleaving metalloprotease ADAMTS13 have emerged as significant targets of the downstream effect of endothelial breakdown and coagulation dysregulation. Elucidation of the mechanism by which the dysregulated VWF-ADAMTS13 axis leads to endothelial dysfunction and coagulopathy after trauma can help identify new targets for therapy and sites for intervention. Much of what is known mechanistically regarding VWF stems from work done in traumatic brain injury. Following localized brain injury, brain-derived extracellular vesicles are released into circulation where they induce a hypercoagulable state that rapidly turns into consumptive coagulopathy. VWF released from injured endothelial cells binds to these extracellular vesicles to enhance their activity in promoting coagulopathy and increasing endothelial permeability. However, there are numerous gaps in our knowledge of VWF following injury, providing a platform for further investigation.

Novel Approaches to Fine-Tune Therapeutic Targeting of Platelets in Atherosclerosis: A Critical Appraisal.

The pathogenesis of atherosclerotic vascular disease is driven by a multitude of risk factors intertwining metabolic and inflammatory pathways. Increasing knowledge about platelet biology sheds light on how platelets take part in these processes from early to later stages of plaque development. Recent insights from experimental studies and mouse models substantiate platelets as initiators and amplifiers in atherogenic leukocyte recruitment. These studies are complemented by results from genetics studies shedding light on novel molecular mechanisms which provide an interesting prospect as novel targets. For instance, experimental studies provide further details how platelet-decorated von Willebrand factor tethered to activated endothelial cells plays a role in atherogenic monocyte recruitment. Novel aspects of platelets as atherogenic inductors of neutrophil extracellular traps and particularities in signaling pathways such as cyclic guanosine monophosphate and the inhibitory adaptor molecule SHB23/LNK associating platelets with atherogenesis are shared. In summary, it was our intention to balance insights from recent experimental data that support a plausible role for platelets in atherogenesis against a paucity of clinical evidence needed to validate this concept in humans.

Acute Kidney Injury in Decompensated Cirrhosis Is Associated With Both Hypo-coagulable and Hyper-coagulable Features.

BACKGROUND AND AIMS: Recent evidence suggests that acute kidney injury (AKI) is the main predictor of postparacentesis bleeding in patients with cirrhosis. To assess the factors responsible for bleeding tendency in AKI, we performed a prospective study comparing all three aspects of hemostasis (platelets, coagulation, and fibrinolysis) in patients with decompensated cirrhosis with and without AKI. APPROACH AND RESULTS: Primary hemostasis assessment included platelet aggregation and secretion (platelet function markers) and von Willebrand factor. Secondary hemostasis assessment included pro-coagulant (factor VIII and factor XIII) and anti-coagulant (protein C, protein S, and antithrombin) factors and thrombin generation. Tertiary hemostasis assessment included fibrinolytic factors and plasmin-antiplasmin complex. Eighty patients with decompensated cirrhosis were recruited (40 each with and without AKI). Severity of cirrhosis and platelet count were comparable between groups. Median serum creatinine was 1.8 mg/dL and 0.8 mg/dL in patients with and without AKI, respectively. At baseline, patients with cirrhosis and AKI had lower platelet aggregation and secretion, indicative of impaired platelet function (increased bleeding tendency), without differences in von Willebrand factor. Regarding coagulation factors, factor VIII was higher, whereas protein C, protein S, and antithrombin were all lower, which, together with increased thrombin generation, indicate hypercoagulability. In contrast, factor XIII was lower in AKI (increased bleeding tendency). Finally, while both hypofibrinolytic and hyperfibrinolytic changes were present in AKI, a higher plasmin-antiplasmin complex indicated a hyperfibrinolytic state. After AKI resolution (n = 23 of 40), platelet function and coagulation improved to levels observed in patients with cirrhosis patients without AKI; however, fibrinolysis remained hyperactivated. CONCLUSIONS: In patients with decompensated cirrhosis, AKI is associated with both hypocoagulable and hypercoagulable features that can potentially increase the risk of both bleeding and thrombosis.

The protective rather than prothrombotic fibrinogen in COVID-19 and other inflammatory states.

Hypercoagulability has been recognized as a common complication of COVID-19. Exact mechanisms for this extreme coagulation activation have not yet been elucidated. However, one of the consistent laboratory finding is the increase in fibrinogen, in some cases, marked elevation. High circulating levels of fibrinogen have been linked to thrombosis for years and for this reason, hyperfibrinogenemia is considered one of the mechanisms for COVID-19 coagulopathy. In this forum article, instead of the prothrombotic role, a protective function for fibrinogen is discussed. Fibrinogen, like the other well-known acute phase reactants, is increased in COVID-19 possibly to protect the host.

Endothelial CD40 Mediates Microvascular von Willebrand Factor-Dependent Platelet Adhesion Inducing Inflammatory Venothrombosis in ADAMTS13 Knockout Mice.

BACKGROUND: von Willebrand factor (vWF) plays an important role in platelet activation. CD40-CD40 ligand (CD40L) induced vWF release has been described in large vessels and cultured endothelium, but its role in the microcirculation is not known. Here, we studied whether CD40 is expressed in murine microvessels in vivo, whether CD40L induces platelet adhesion and leukocyte activation, and how deficiency of the vWF cleaving enzyme ADAMTS13 affects these processes. METHODS AND RESULTS: The role of CD40L in the formation of beaded platelet strings reflecting their adhesion to ultralarge vWF fibers (ULVWF) was analyzed in the murine cremaster microcirculation in vivo. Expression of CD40 and vWF was studied by immunohistochemistry in isolated and fixed cremasters. Microvascular CD40 was only expressed under inflammatory conditions and exclusively in venous endothelium. We demonstrate that CD40L treatment augmented the number of platelet strings, reflecting ULVWF multimer formation exclusively in venules and small veins. In ADAMTS13 knockout mice, the number of platelet strings further increased to a significant extent. As a consequence extensive thrombus formation was induced in venules of ADAMTS13 knockout mice. In addition, circulating leukocytes showed primary and rapid adherence to these platelet strings followed by preferential extravasation in these areas. CONCLUSION: CD40L is an important stimulus of microvascular endothelial ULVWF release, subsequent platelet string formation and leukocyte extravasation but only in venous vessels under inflammatory conditions. Here, the lack of ADAMTS13 leads to severe thrombus formation. The results identify CD40 expression and ADAMTS13 activity as important targets to prevent microvascular inflammatory thrombosis.

Functional regulation of von Willebrand factor ameliorates acute ischemia-reperfusion kidney injury in mice.

Acute kidney injury (AKI), an abrupt loss of renal function, is often seen in clinical settings and may become fatal. In addition to its hemostatic functions, von Willebrand factor (VWF) is known to play a role in cross-talk between inflammation and thrombosis. We hypothesized that VWF may be involved in the pathophysiology of AKI, major causes of which include insufficient renal circulation or inflammatory cell infiltration in the kidney. To test this hypothesis, we studied the role of VWF in AKI using a mouse model of acute ischemia-reperfusion (I/R) kidney injury. We analyzed renal function and blood flow in VWF-gene deleted (knock-out; KO) mice. The functional regulation of VWF by ADAMTS13 or a function-blocking anti-VWF antibody was also evaluated in this pathological condition. Greater renal blood flow and lower serum creatinine were observed after reperfusion in VWF-KO mice compared with wild-type (WT) mice. Histological analysis also revealed a significantly lower degree of tubular damage and neutrophil infiltration in kidney tissues of VWF-KO mice. Both human recombinant ADAMTS13 and a function-blocking anti-VWF antibody significantly improved renal blood flow, renal function and histological findings in WT mice. Our results indicate that VWF plays a role in the pathogenesis of AKI. Proper functional regulation of VWF may improve the microcirculation and vessel function in the kidney, suggesting a novel therapeutic option against AKI.

Apolipoprotein E4 Expression Causes Gain of Toxic Function in Isogenic Human Induced Pluripotent Stem Cell-Derived Endothelial Cells.

OBJECTIVE: The ApoE (apolipoprotein) allele epsilon 4 is a major genetic risk factor for Alzheimer disease, cardiovascular disorders, and stroke, indicating that it significantly impacts cerebral and vascular systems. However, very little is known about how APOE genotype affects brain endothelial cells, which form a network of tight junctions to regulate communication between the brain and circulating blood factors. Approach and Results: Here, we present a novel model of endothelial dysfunction using isogenic human induced pluripotent stem cell-derived cells harboring different alleles of the APOE gene, specifically ApoE 3/3, 3/4, and 4/4. We show for the first time that ApoE4 expression by endothelial cells is sufficient to cause a toxic gain of cellular dysfunction. Using RNAseq, we found significant effects of ApoE4 on signaling pathways involved in blood coagulation and barrier function. These changes were associated with altered cell function, including increased binding of platelets to ECs with the 3/4 or 4/4 genotype. ApoE4-positive cells exhibited a proinflammatory state and prothrombotic state, evidenced by higher secretion of Aß (amyloid-ß) 40 and 42, increased release of cytokines, and overexpression of the platelet-binding protein VWF (vonWillebrand factor). Immunohistochemistry of human brain Alzheimer disease brains also showed increased VWF expression with the ApoE4/4 genotype. Finally, pharmacological inhibition of inflammation in ECs by celastrol rescued overexpression of VWF in cells expressing ApoE4. CONCLUSIONS: These cells provide novel insight into ApoE4-mediated endothelial dysfunction and provide a new platform to test potential therapies for vascular disorders.

Formation and Resolution of Pial Microvascular Thrombosis in a Mouse Model of Thrombotic Thrombocytopenic Purpura.

OBJECTIVE: Microvascular thrombosis is the hallmark pathology of thrombotic thrombocytopenic purpura (TTP), a rare life-threatening disease. Neurological dysfunction is present in over 90% of patients with TTP, and TTP can cause long-lasting neurological damage or death. However, the pathophysiology of microvascular thrombosis in the brain is not well studied to date. Here, we investigate the formation and resolution of thrombosis in pial microvessels. Approach and Results: Using a cranial intravital microscopy in well-established mouse models of congenital TTP induced by infusion of recombinant VWF (von Willebrand factor), we found that soluble VWF, at high concentration, adheres to the endothelium of the vessel wall, self-associates, and initiates platelet adhesion resulting in the formation of pial microvascular thrombosis in ADAMTS13-/- (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) mice. Importantly, VWF-mediated pial microvascular thrombosis occurred without vascular injury to the brain, and thrombi consisted of resting platelets adhered onto ultra-large VWF without fibrin in the brain in rVWF (recombinant VWF) challenged ADAMTS13-/- mice. Prophylactic treatment with recombinant ADAMTS13 (BAX930) effectively prevented the onset of the VWF-mediated microvascular thrombosis and therapeutic treatment with BAX930 acutely resolved the preexisting or growing thrombi in the brain of ADAMTS13-/- mice after rVWF challenge. The absence of platelet activation and fibrin formation within VWF-mediated thrombi and efficacy of BAX930 was confirmed with an endothelial-driven VWF-mediated microvascular thrombosis model in mice. CONCLUSIONS: Our results provide important insight into the initiation and development of microvascular thrombi in mouse models that mimics TTP and indicate that rADAMTS13 could be an effective interventional therapy for microvascular thrombosis, the hallmark pathology in TTP.

Turbulent Flow Promotes Cleavage of VWF (von Willebrand Factor) by ADAMTS13 (A Disintegrin and Metalloproteinase With a Thrombospondin Type-1 Motif, Member 13).

Objective- Acquired von Willebrand syndrome is defined by excessive cleavage of the VWF (von Willebrand Factor) and is associated with impaired primary hemostasis and severe bleeding. It often develops when blood is exposed to nonphysiological flow such as in aortic stenosis or mechanical circulatory support. We evaluated the role of laminar, transitional, and turbulent flow on VWF cleavage and the effects on VWF function. Approach and Results- We used a vane rheometer to generate laminar, transitional, and turbulent flow and evaluate the effect of each on VWF cleavage in the presence of ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type-1 motif, member 13). We performed functional assays to evaluate the effect of these flows on VWF structure and function. Computational fluid dynamics was used to estimate the flow fields and forces within the vane rheometer under each flow condition. Turbulent flow is required for excessive cleavage of VWF in an ADAMTS13-dependent manner. The assay was repeated with whole blood, and the turbulent flow had the same effect. Our computational fluid dynamics results show that under turbulent conditions, the Kolmogorov scale approaches the size of VWF. Finally, cleavage of VWF in this study has functional consequences under flow as the resulting VWF has decreased ability to bind platelets and collagen. Conclusions- Turbulent flow mediates VWF cleavage in the presence of ADAMTS13, decreasing the ability of VWF to sustain platelet adhesion. These findings impact the design of mechanical circulatory support devices and are relevant to pathological environments where turbulence is added to circulation.

Pre-procedural abnormal function of von Willebrand Factor is predictive of bleeding after surgical but not transcatheter aortic valve replacement.

Both transcatheter aortic valve implantation (TAVI) and surgical aortic valve replacement (SAVR) have been proven to effectively correct von Willebrand Factor (vWF) pathologies, however there is limited data simultaneously comparing outcomes of both approaches. We prospectively enrolled patients with severe aortic stenosis referred for TAVI (n = 52) or SAVR (n = 48). In each case, vWF antigen (vWF:Ag), vWF activity (vWF:Ac) and activity-to-antigen (vWF:Ac/Ag) ratio were assessed at baseline, 24 h and 72 h after procedure. VWF abnormalities were defined as reduced vWF:Ac/Ag ratio (< 0.8). Bleeding events in both arms were classified according to Valve Academic Research Consortium (VARC-2) definitions. Overall, there was no difference between patients referred for TAVI and SAVR in vWF:Ac (1.62 ± 0.52 vs 1.71 ± 0.64; p = 0.593), vWF:Ag (1.99 ± 0.81 vs 2.04 ± 0.81; p = 0.942) or vWF:Ac/Ag ratio (0.84 ± 0.16 vs 0.85 ± 0.12; p = 0.950). Pathological vWF:Ac/Ag ratio was found in 20 (38%) TAVI and 15 (31%) SAVR patients (p = 0.407). Normalization of vWF:Ac/Ag ratio at day 3 after procedure was achieved in 19 (95%) TAVI and 13 (87%) SAVR patients (p = 0.439). Similar prevalence of major or life-threatening bleedings (MLTB) after TAVI and SAVR in entire groups was observed (19% vs. 23%, p = 0.652). VWF abnormalities were associated with higher incidence of MLTB in SAVR (53% vs 9%, p < 0.001), but not TAVI (15% vs. 22%, p = 0.132). Accordingly, in receiver-operating characteristic curve analysis vWF:Ac/Ag ratio < 0.8 showed significant sensitivity and specificity for predicting MLTB in SAVR group (AUC 0.735, 95% CI 0.538-0.931, p = 0.019). We proved that abnormal function of vWF is corrected successfully after both TAVI and SAVR, but vWF abnormalities are predictive of MLTB only in surgical patients.

Desialylation of platelets induced by Von Willebrand Factor is a novel mechanism of platelet clearance in dengue.

Thrombocytopenia and platelet dysfunction are commonly observed in patients with dengue virus (DENV) infection and may contribute to complications such as bleeding and plasma leakage. The etiology of dengue-associated thrombocytopenia is multifactorial and includes increased platelet clearance. The binding of the coagulation protein von Willebrand factor (VWF) to the platelet membrane and removal of sialic acid (desialylation) are two well-known mechanisms of platelet clearance, but whether these conditions also contribute to thrombocytopenia in dengue infection is unknown. In two observational cohort studies in Bandung and Jepara, Indonesia, we show that adult patients with dengue not only had higher plasma concentrations of plasma VWF antigen and active VWF, but that circulating platelets had also bound more VWF to their membrane. The amount of platelet-VWF binding correlated well with platelet count. Furthermore, sialic acid levels in dengue patients were significantly reduced as assessed by the binding of Sambucus nigra lectin (SNA) and Maackia amurensis lectin II (MAL-II) to platelets. Sialic acid on the platelet membrane is neuraminidase-labile, but dengue virus has no known neuraminidase activity. Indeed, no detectable activity of neuraminidase was present in plasma of dengue patients and no desialylation was found of plasma transferrin. Platelet sialylation was also not altered by in vitro exposure of platelets to DENV nonstructural protein 1 or cultured DENV. In contrast, induction of binding of VWF to glycoprotein 1b on platelets using the VWF-activating protein ristocetin resulted in the removal of platelet sialic acid by translocation of platelet neuraminidase to the platelet surface. The neuraminidase inhibitor oseltamivir reduced VWF-induced platelet desialylation. Our data demonstrate that excessive binding of VWF to platelets in dengue results in neuraminidase-mediated platelet desialylation and platelet clearance. Oseltamivir might be a novel treatment option for severe thrombocytopenia in dengue infection.

von Willebrand factor deficiency does not influence angiotensin II-induced abdominal aortic aneurysm formation in mice.

Abdominal aortic aneurysm (AAA) refers to a localized dilation of the abdominal aorta that exceeds the normal diameter by 50%. AAA pathophysiology is characterized by progressive inflammation, vessel wall destabilization and thrombus formation. Our aim was to investigate the potential involvement of von Willebrand factor (VWF), a thrombo-inflammatory plasma protein, in AAA pathophysiology using a dissection-based and angiotensin II infusion-induced AAA mouse model. AAA formation was induced in both wild-type and VWF-deficient mice by subcutaneous implantation of an osmotic pump, continuously releasing 1000 ng/kg/min angiotensin II. Survival was monitored, but no significant difference was observed between both groups. After 28 days, the suprarenal aortic segment of the surviving mice was harvested. Both AAA incidence and severity were similar in wild-type and VWF-deficient mice, indicating that AAA formation was not significantly influenced by the absence of VWF. Although VWF plasma levels increased after the infusion period, these increases were not correlated with AAA progression. Also detailed histological analyses of important AAA hallmarks, including elastic degradation, intramural thrombus formation and leukocyte infiltration, did not reveal differences between both groups. These data suggest that, at least in the angiotensin II infusion-induced AAA mouse model, the role of VWF in AAA pathophysiology is limited.

Tuning the endothelial response: differential release of exocytic cargos from Weibel-Palade bodies.

Essentials Endothelial activation initiates multiple processes, including hemostasis and inflammation. The molecules that contribute to these processes are co-stored in secretory granules. How can the cells control release of granule content to allow differentiated responses? Selected agonists recruit an exocytosis-linked actin ring to boost release of a subset of cargo. SUMMARY: Background Endothelial cells harbor specialized storage organelles, Weibel-Palade bodies (WPBs). Exocytosis of WPB content into the vascular lumen initiates primary hemostasis, mediated by von Willebrand factor (VWF), and inflammation, mediated by several proteins including P-selectin. During full fusion, secretion of this large hemostatic protein and smaller pro-inflammatory proteins are thought to be inextricably linked. Objective To determine if secretagogue-dependent differential release of WPB cargo occurs, and whether this is mediated by the formation of an actomyosin ring during exocytosis. Methods We used VWF string analysis, leukocyte rolling assays, ELISA, spinning disk confocal microscopy, high-throughput confocal microscopy and inhibitor and siRNA treatments to demonstrate the existence of cellular machinery that allows differential release of WPB cargo proteins. Results Inhibition of the actomyosin ring differentially effects two processes regulated by WPB exocytosis; it perturbs VWF string formation but has no effect on leukocyte rolling. The efficiency of ring recruitment correlates with VWF release; the ratio of release of VWF to small cargoes decreases when ring recruitment is inhibited. The recruitment of the actin ring is time dependent (fusion events occurring directly after stimulation are less likely to initiate hemostasis than later events) and is activated by protein kinase C (PKC) isoforms. Conclusions Secretagogues differentially recruit the actomyosin ring, thus demonstrating one mechanism by which the prothrombotic effect of endothelial activation can be modulated. This potentially limits thrombosis whilst permitting a normal inflammatory response. These results have implications for the assessment of WPB fusion, cargo-content release and the treatment of patients with von Willebrand disease.