Support Platelet Aggregation Research Articles

Hydroxychloroquine inhibits hemolysis-induced arterial thrombosis ex vivo and improves lung perfusion in hemin-treated mice

BackgroundFree labile hemin acts as a damage-associated molecular pattern during acute and chronic hemolysis and muscle injury, supporting platelet activation and thrombosis. ObjectivesTo investigate the anti-thrombotic potential of hydroxychloroquine on hemolysis-induced platelet activation and arterial thrombosis. MethodsThe effect of hydroxychloroquine on hemin-induced platelet activation and hemolysis-induced platelet recruitment and aggregation was measured in washed platelets and hemolyzed blood, respectively. Its effect on ferric-chloride (FeCl3)-induced arterial thrombosis and lung perfusion following hemin injection was assessed in wild-type mice. ResultsErythrocyte lysis and endothelial cell activation cooperatively supported platelet aggregation and thrombosis at arterial shear stress. This thrombotic effect was reversed by hydroxychloroquine. In a purified system, hydroxychloroquine inhibited platelet build-up on immobilized von Willebrand factor in hemolyzed blood without altering initial platelet recruitment. Hydroxychloroquine inhibited hemin-induced platelet activation and phosphatidylserine exposure independently of reactive oxygen species generation. In the presence of hemin, hydroxychloroquine did not alter glycoprotein VI shedding but reduced C-type-lectin-like-2 expression on platelets. In vivo, hydroxychloroquine reversed pulmonary perfusion decline induced by exogenous administration of hemin. In arterial thrombosis models, hydroxychloroquine inhibited ferric-chloride–induced thrombosis in the carotid artery and reduced von Willebrand factor accumulation in the thrombi. ConclusionHydroxychloroquine inhibited hemolysis-induced arterial thrombosis ex vivo and improved pulmonary perfusion in hemin-treated mice, supporting a potential benefit of its use as an adjuvant therapy in hemolytic diseases to limit arterial thrombosis and to improve organ perfusion.

CLEC-2 Supports Platelet Aggregation in Mouse but not Human Blood at Arterial Shear.

C-type lectin-like receptor 2 (CLEC-2) is highly expressed on platelets and a subpopulation of myeloid cells, and is critical in lymphatic development. CLEC-2 has been shown to support thrombus formation at sites of inflammation, but to have a minor/negligible role in hemostasis. This identifies CLEC-2 as a promising therapeutic target in thromboinflammatory disorders, without hemostatic detriment. We utilized a GPIbα-Cre recombinase mouse for more restricted deletion of platelet-CLEC-2 than the previously used PF4-Cre mouse. clec1bfl/flGPIbα-Cre+ mice are born at a Mendelian ratio, with a mild reduction in platelet count, and present with reduced thrombus size post-FeCl3-induced thrombosis, compared to littermates. Antibody-mediated depletion of platelet count in C57BL/6 mice, to match clec1bfl/flGPIbα-Cre+ mice, revealed that the reduced thrombus size post-FeCl3-injury was due to the loss of CLEC-2, and not mild thrombocytopenia. Similarly, clec1bfl/flGPIbα-Cre+ mouse blood replenished with CLEC-2-deficient platelets ex vivo to match littermates had reduced aggregate formation when perfused over collagen at arterial flow rates. In contrast, platelet-rich thrombi formed following perfusion of human blood under flow conditions over collagen types I or III, atherosclerotic plaque, or inflammatory endothelial cells were unaltered in the presence of CLEC-2-blocking antibody, AYP1, or recombinant CLEC-2-Fc. The reduction in platelet aggregation observed in clec1bfl/flGPIbα-Cre+ mice during arterial thrombosis is mediated by the loss of CLEC-2 on mouse platelets. In contrast, CLEC-2 does not support thrombus generation on collagen, atherosclerotic plaque, or inflamed endothelial cells in human at arterial shear.

Alpha-Dystroglycan Supports Platelet Aggregation and Thrombus Formation

No relevant conflicts of interest to declare.

Greetings from the endoplasmic reticulum (ER): escaping ER thiol isomerases regulate thrombosis

Greetings from the endoplasmic reticulum (ER): escaping ER thiol isomerases regulate thrombosis

αIIbβ3 binding to a fibrinogen fragment lacking the γ-chain dodecapeptide is activation dependent and EDTA inducible

AbstractPlatelet integrin receptor αIIbβ3 supports platelet aggregation by binding fibrinogen. The interaction between the fibrinogen C-terminal γ-chain peptide composed of residues γ-404-411 (GAKQAGDV) and the Arg-Gly-Asp (RGD) binding pocket on αIIbβ3 is required for fibrinogen-mediated platelet aggregation, but data suggest that other ancillary binding sites on both fibrinogen and αIIbβ3 may lead to higher-affinity fibrinogen binding and clot retraction. To identify additional sites, we analyzed the ability of platelets and cells expressing normal and mutant αIIbβ3 to adhere to an immobilized fibrinogen plasmin fragment that lacks intact γ-404-411 ('D98'). We found the following: (1) Activated, but not unactivated, platelets adhere well to immobilized 'D98.' (2) Cells expressing constitutively active αIIbβ3 mutants, but not cells expressing normal αIIbβ3 or αVβ3, adhere well to 'D98.' (3) Monoclonal antibodies 10E5 and 7E3 inhibit the adhesion to 'D98' of activated platelets and cells expressing constitutively active αIIbβ3, as do small-molecule inhibitors that bind to the RGD pocket. (4) EDTA paradoxically induces normal αIIbβ3 to interact with 'D98.' Because molecular modeling and molecular dynamics simulations suggested that the αIIb L151-D159 helix may contribute to the interaction with 'D98,' we studied an αIIbβ3 mutant in which the αIIb 148-166 loop was swapped with the corresponding αV loop; it failed to bind to fibrinogen or 'D98.' Our data support a model in which conformational changes in αIIbβ3 and/or fibrinogen after platelet activation and the interaction between γ-404-411 and the RGD binding pocket make new ancillary sites available that support higher-affinity fibrinogen binding and clot retraction.

The Second CGHC Motif of Protein Disulfide Isomerase Mediates Thrombosis

Protein disulfide isomerase (PDI) catalyzes thiol-disulfide exchange reactions that are critical for protein function. PDI has two distinct CGHC redox-active sites, but nothing is known about which site is important in physiologic reactions including thrombosis. To study the role of PDI and its active sites in thrombosis we used two genetically-modified models, Mx1-Cre/PDIfl/fl mice with blood cells and vessel wall cells lacking PDI, and transgenic PDI(ss-oo) mice with PDI lacking the second CGHC active site. Using laser-induced cremaster arteriole injury fibrin deposition was decreased in both mouse models. Both mouse models showed attenuation of platelet accumulation using laser arterial injury and FeCl3-induced mesenteric artery injury. The defects in fibrin deposition and platelet accumulation were rescued by infusion of recombinant PDI(oo-ss) containing only a functional second active site, confirming the second active site of PDI was critical for both fibrin formation and platelet accumulation. In vitro studies showed that platelet aggregation depended on the second CGHC motif of PDI, and that binding of PDI protein to platelets depended on the presence of the αIIbβ3 integrin. Studies on platelet secretion revealed that P-selectin expression and ATP release were dependent on a functional second active site of PDI. Treatment of the platelets with eptifibatide did not affect P-selectin expression. Together these findings suggest the second active of PDI catalyzes a reaction in αIIbβ3 supporting platelet aggregation and a reaction in a non-αIIbβ3 substrate supporting secretion. In vivo studies on coagulation showed that PDI infusion rescued fibrin formation in eptifibatide-treated mice in which platelet accumulation was completely blocked, suggesting that PDI directly contributes to activation of coagulation. In summary, our results indicate that the second CGHC active site of PDI has a role in platelet function and coagulation. Targeting this site may have a dual inhibitory effect on thrombosis by decreasing both platelet and fibrin accumulation. DisclosuresNo relevant conflicts of interest to declare.

Development of a Hyperactive Primary Hemostatic System During Off-Pump Lung Transplantation Resulting From an Unbalance Between von Willebrand Factor and Its Cleaving Protease ADAMTS13

An unbalance between the platelet-adhesive protein von Willebrand factor (VWF) and its cleaving protease ADAMTS13 is a risk factor for thrombosis. Here, we assessed levels and functionality of VWF and ADAMTS13 in patients undergoing off-pump lung transplantation. We analyzed plasma of 10 patients and distinguished lung transplantation-specific effects from those generally accompanying open-chest surgeries by comparing results with 11 patients undergoing off-pump coronary bypass graft (CABG) surgery. Forty healthy volunteers were included for reference values. VWF antigen levels as well as the VWF ristocetin cofactor activity/VWF antigen ratio increased during lung transplantation and after CABG surgery. An increase in VWF propeptide levels was paralleled by a decrease in ADAMTS13 activity. This was more pronounced during lung transplantation. Similarly, the capacity of plasma to support platelet aggregation under shear flow conditions in vitro was more increased during lung transplantation. The proportion of high molecular weight VWF multimers was elevated in both groups without evidence for ultra-large VWF. VWF's collagen binding activity remained unchanged. In conclusion, a hyperactive primary hemostatic system develops during lung transplantation resulting both from a pronounced (functional) increase of the VWF molecule and decrease of ADAMTS13. This may increase the risk of platelet thrombosis within the allograft.

Integrin αIIbβ3-Mediated Outside-in Signaling: Brake or Amplifier of Platelet Activation?

αIIbβ3 is the most prominent integrin in platelets, and binding to its ligands, in addition to supporting platelet aggregation, also results in the transmission of so-called αIIbβ3-mediated outside-in signals into the cell interior. While it is well accepted that integrin-mediated outside-in signaling functions as an amplifier of platelet activation, accumulating evidence suggests that outside-in signaling can, under certain conditions, function as an inhibitor of platelet activation. In this regard, previous studies have shown that ligand binding and platelet aggregation activate the inositol phosphatase SHIP-1, a negative regulator of the PI3K/Akt signaling pathway, to shift activated integrins back to their resting state, leading to dissociation of platelet aggregates. Because the PI3K/Akt signaling pathway is also involved in platelet granule secretion, we examined whether ligand binding to αIIbβ3 might transmit inhibitory signals that suppress platelet granule secretion. Interestingly, we found that antagonists of integrin αIIbβ3 promote both platelet dense- and α-granule secretion stimulated by low dose agonists. In support of this finding, both mouse and human platelets lacking expression of αIIbβ3 exhibited increased granule secretion compared to their wild-type counterparts. Conversely, Mn++-induced fibrinogen binding to αIIbβ3 inhibited low-dose agonist-induced platelet granule secretion. Biochemical analysis revealed that blocking ligand binding to, or absence of, αIIbβ3, enhanced agonist-induced Akt phosphorylation, while at the same time prevented the activation of the inhibitory enzyme, SHIP-1. To further investigate the role of SHIP-1 in inhibitory signaling, we examined the effect on platelet secretion of 3AC, a specific inhibitor of SHIP-1. We found that 3AC not only restores ADP-induced platelet granule secretion, but also increases CRP- or TRAP-induced platelet granule secretion. Taken together, these data demonstrate that integrin αIIbβ3-mediated outside-in signaling act as a brake to restrict unnecessary platelet activation that occurs in the presence of low-dose agonist stimulation. DisclosuresNo relevant conflicts of interest to declare.

Folic Acid‐Conjugated 4‐Amino‐Phenylboronate, a Boron‐Containing Compound Designed for Boron Neutron Capture Therapy, is an Unexpected Agonist for Human Neutrophils and Platelets

Boron neutron capture therapy (BNCT) is an anticancer treatment based on the accumulation in the tumor cells of (10) B-containing molecules and subsequent irradiation with low-energy neutrons, which bring about the decay of (10) B to very toxic (7) Li(3+) and (4) He(2+) ions. The effectiveness of BNCT is limited by the low delivery and accumulation of the used (10) B-containing compounds. Here, we report the development of folic acid-conjugated 4-amino-phenylboronate as a novel possible compound for the selective delivery of (10) B in BNCT. An extensive analysis about its biocompatibility to mature blood cells and platelet progenitors revealed that the compound markedly supports platelet aggregation, neutrophil oxidative burst, and inhibition of megakaryocyte development, while it does not have any manifest effect on red blood cells.

Impaired thrombin-induced platelet activation and thrombus formation in mice lacking the Ca2+-dependent tyrosine kinase Pyk2

AbstractIn the present study, we used a knockout murine model to analyze the contribution of the Ca2+-dependent focal adhesion kinase Pyk2 in platelet activation and thrombus formation in vivo. We found that Pyk2-knockout mice had a tail bleeding time that was slightly increased compared with their wild-type littermates. Moreover, in an in vivo model of femoral artery thrombosis, the time to arterial occlusion was significantly prolonged in mice lacking Pyk2. Pyk2-deficient mice were also significantly protected from collagen plus epinephrine-induced pulmonary thromboembolism. Ex vivo aggregation of Pyk2-deficient platelets was normal on stimulation of glycoprotein VI, but was significantly reduced in response to PAR4-activating peptide, low doses of thrombin, or U46619. Defective platelet aggregation was accompanied by impaired inside-out activation of integrin αIIbβ3 and fibrinogen binding. Granule secretion was only slightly reduced in the absence of Pyk2, whereas a marked inhibition of thrombin-induced thromboxane A2 production was observed, which was found to be responsible for the defective aggregation. Moreover, we have demonstrated that Pyk2 is implicated in the signaling pathway for cPLA2 phosphorylation through p38 MAPK. The results of the present study show the importance of the focal adhesion kinase Pyk2 downstream of G-protein–coupled receptors in supporting platelet aggregation and thrombus formation.

Platelet activity and cardiovascular risk in apparently healthy individuals: a review of the data

Cardiovascular disease is a major cause of morbidity and mortality. Numerous risk scores exist to identify healthy individuals at increased risk of developing cardiovascular disease. Although platelets are a key mediator in the pathogenesis of cardiovascular disease, the role of platelet activity measurements and the incidence of cardiovascular disease are uncertain. Platelet aggregometry-the most well studied method of platelet function testing-is associated with risk factors for cardiovascular disease. However, data supporting platelet aggregation and incident cardiovascular disease is conflicting. Plasma markers of platelet activation are promising candidates. Soluble CD40L and P-selectin are easily measured with a standardized ELISA, and there is some data to suggest an association with cardiovascular disease, but further studies are required. While mean platelet volume is a promising candidate, platelet count and bleeding time are not specific for platelet activity nor are they associated with cardiovascular disease in a healthy population. For this field to progress, we recommend large-scale, prospective studies that measure a battery of these platelet function tests in individuals without cardiovascular disease to better understand the associations, if any, between platelet activity and cardiovascular disease.

Plasma Fibronectin In Thrombosis and Hemostasis: Exploring the Fibrin Dependent and Independent Mechanisms

AbstractAbstract 484 Background:Plasma fibronectin (pFn) is an abundant protein in the blood. It has long been suspected that pFn plays a role in thrombosis/hemostasis, but this has remained controversial. Our previous study using pFn deficient mice demonstrated that pFn supports thrombosis (PNAS. 2003; 100: 2415-9). Unexpectedly, depletion of pFn in fibrinogen (Fg) and von Willebrand factor (VWF) double deficient (Fg/VWF−/−) mice enhanced, rather than abolished, platelet aggregation and thrombus formation, revealing a functional switch of pFn in the presence and absence of Fg and VWF (Blood. 2009;113:1809-17). However, the mechanism that controls this switch is not known. Furthermore, the hemostatic function of pFn in VW disease (VWD) or afibrinogenemia is unclear. Methods:To address these questions, we bred pFn conditional knockout mice with VWF−/− or Fg−/− mice, establishing 2 new strains of mice: Fg/pFn−/− and VWF/pFn−/−. We also extended our studies of pFn in the triple knockout (TKO, Fg/VWF/pFn−/−) mice. PolyI-polyC was injected into Cre+ and Cre- mice, which resulted in the depletion of plasma pFn (>98%) and platelet pFn (>80%) in Cre+ mice but not in Cre- littermate controls. Aggregometry, a perfusion chamber system, thromboelastography (TEG), tail vein bleeding assay and intravital microscopy were used to study these mice. Results:We first observed a significantly higher mortality in TKO (25%, P<0.05) mice than their Cre- Fg/VWF−/− littermates within 1–2 weeks following the depletion of pFn. Autopsy of these mice revealed severe subcutaneous or abdominal bleeding at the sites of injection. The tail vein bleeding time in TKO mice was also prolonged (P<0.05). Using a laser injury model of intravital microscopy, we observed rapid deposition of fluorescently-labeled pFn at sites of vessel injury in Fg/VWF−/− mice prior to significant platelet deposition. This suggested pFn is a quick/efficient factor contributing to hemostasis in the absence of Fg/VWF.We further found that the mortality rate in Fg/pFn−/− mice was also higher than their Cre- Fg−/− littermates (29%, P<0.05), demonstrating that pFn is a critical hemostatic factor that prevents fatal hemorrhage in afibrinogenemic mice via a fibrin-independent mechanism.We also found that pFn supports hemostasis in VWF−/− mice, although no significant mortality difference was observed (P>0.05). The tail vein bleeding time was longer in VWF/pFn−/− mice than in Cre- VWF−/− littermates (P<0.05), and significantly smaller thrombi were observed when VWF/pFn−/− whole blood was perfused over a collagen surface under shear rate of 1800s-1 (P<0.05). This suggests that pFn may play a role in VWF deficiency (i.e. in type 3 VWD).pFn was also found to support hemostasis in a fibrin-dependent manner. We first demonstrated with TEG that fibrin clot strength was significantly stronger in Cre- littermates than in pFn−/− mice (P<0.05). Platelet aggregation in gel-filtered platelets induced by thrombin, which converts Fg to fibrin on the platelet surface, was greater in Cre- VWF−/− than VWF/pFn−/− platelets (P<0.05).Very interestingly, in keeping with our earlier observation in TKO mice, pFn also inhibited platelet aggregation when fibrin was absent. In Fg−/− mice, we found that pFn depletion enhanced gel-filtered platelet aggregation induced by both thrombin and thrombin receptor activating peptide (TRAP, AYPGKF; P<0.05). In Cre- VWF−/− mice where Fg is present, pFn depletion also enhanced gel-filtered platelet aggregation induced by TRAP (which cannot convert Fg to fibrin) (P<0.05). pFn therefore plays a dual role in platelet aggregation based on the presence of fibrin (i.e. covalently linked fibrin-pFn supports platelet aggregation, while pFn alone inhibits aggregation). Conclusion:Our data demonstrated that pFn is a critical factor for the survival of Fg−/− mice and supports hemostasis in VWF−/− mice via both fibrin-independent and dependent pathways. We clearly showed that fibrin, likely in the form of covalently-linked fibrin-pFn complexes, is required for pFn to support platelet aggregation. Through inhibition of platelet aggregation, non-fibrin-linked soluble pFn may play an important role in the prevention of excessive thrombus formation at the site of vessel injury and thus maintains blood circulation. pFn is therefore likely a crucial supportive factor in hemostasis (for afibrinogenemic and VWD patients), and an important regulator in thrombosis. Disclosures:No relevant conflicts of interest to declare.

Platelet Activation and Biofilm Formation by Aerococcus urinae , an Endocarditis-Causing Pathogen

The Gram-positive bacterium Aerococcus urinae can cause infectious endocarditis (IE) in older persons. Biofilm formation and platelet aggregation are believed to contribute to bacterial virulence in IE. Five A. urinae isolates from human blood were shown to form biofilms in vitro, and biofilm formation was enhanced by the presence of human plasma. Four of the A. urinae isolates caused platelet aggregation in platelet-rich plasma from healthy donors. The Au3 isolate, which induced platelet aggregation in all donors, also activated platelets, as determined by flow cytometry. Platelet aggregation was dependent on bacterial protein structures and on platelet activation since it was sensitive to both trypsin and prostaglandin E(1). Plasma proteins at the bacterial surface were needed for platelet aggregation; and roles of the complement system, fibrinogen, and immunoglobulin G were demonstrated. Complement-depleted serum was unable to support platelet aggregation by Au3 and complement blockade using compstatin-inhibited platelet activation. Platelet activation by Au3 was inhibited by blocking of the platelet fibrinogen receptor, and this isolate was also shown to bind to radiolabeled fibrinogen. Removal of IgG from platelet-rich plasma by a specific protease inhibited the platelet aggregation induced by A. urinae, and blockade of the platelet FcRγIIa hindered platelet activation induced by Au3. Convalescent-phase serum from a patient with A. urinae IE transferred the ability of the bacterium to aggregate platelets in an otherwise nonresponsive donor. Our results show that A. urinae exhibits virulence strategies of importance for IE.

Identification of novel downstream targets of platelet glycoprotein VI activation by differential proteome analysis: implications for thrombus formation

AbstractPlatelets play a key role in hemostasis and various diseases including arterial thrombosis. Glycoprotein VI (GPVI) mediates adhesion to collagen structures exposed at sites of vascular injury and subsequent platelet activation. We determined the effects of specific activation of GPVI on the human platelet proteome. Isolated human platelets were stimulated with an activating monoclonal antibody specific for GPVI. Platelet proteins were analyzed by 2-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry. We identified 8 differentially abundant proteins associated with cell signaling, metabolism, organization and rearrangement of the cytoskeleton, and membrane trafficking. Differentially abundant proteins included aldose reductase (AR), beta-centractin, charged multivesicular body protein 3, Src substrate cortactin, ERp57, and pleckstrin. Importantly, GPVI-modulated protein abundance was functionally relevant. Correspondingly, AR enzyme activity significantly increased upon GPVI activation and inhibition of AR resulted in reduced platelet aggregation. Furthermore, ERp57 was released upon ligation of platelet GPVI and increased the activity of tissue factor, a major initiator of blood coagulation. In summary, GPVI activation results in differential changes in abundance of platelet proteins, including AR and ERp57, which support platelet aggregation and platelet-dependent coagulation. These results provide further insight into the mechanisms that underlie platelet activation through the GPVI receptor and may help to identify novel pharmacologic targets.

Von Willebrand disease and other bleeding disorders in women: consensus on diagnosis and management from an international expert panel

Reproductive tract bleeding in women is a naturally occurring event during menstruation and childbirth. In women with menorrhagia, however, congenital bleeding disorders historically have been underdiagnosed. This consensus is intended to allow physicians to better recognize bleeding disorders as a cause of menorrhagia and consequently offer effective disease-specific therapies.

Collagen-mimetic peptides mediate flow-dependent thrombus formation by high- or low-affinity binding of integrin alpha2beta1 and glycoprotein VI.

Collagen acts as a potent surface for platelet adhesion and thrombus formation under conditions of blood flow. Studies using collagen-derived triple-helical peptides have identified the GXX'GER motif as an adhesive ligand for platelet integrin alpha2beta1, and (GPO)(n) as a binding sequence for the signaling collagen receptor, glycoprotein VI (GPVI). The potency was investigated of triple-helical peptides, consisting of GXX'GER sequences within (GPO)(n) or (GPP)(n) motifs, to support flow-dependent thrombus formation. At a high-shear rate, immobilized peptides containing both the high-affinity alpha2beta1-binding motif GFOGER and the (GPO)(n) motif supported platelet aggregation and procoagulant activity, even in the absence of von Willebrand factor (VWF). With peptides containing only one of these motifs, co-immobilized VWF was needed for thrombus formation. The (GPO)(n) but not the (GPP)(n) sequence induced GPVI-dependent platelet aggregation and procoagulant activity. Peptides with intermediate affinity (GLSGER, GMOGER) or low-affinity (GASGER, GAOGER) alpha2beta1-binding motifs formed procoagulant thrombi only if both (GPO)(n) and VWF were present. At a low-shear rate, immobilized peptides with high- or low-affinity alpha2beta1-binding motifs mediated formation of thrombi with procoagulant platelets only in combination with (GPO)(n). Triple-helical peptides with specific receptor-binding motifs mimic the properties of native collagen I in thrombus formation by binding to both platelet collagen receptors. At a high-shear rate, either GPIb or high-affinity (but not low-affinity) GXX'GER mediates GPVI-dependent formation of procoagulant thrombi. By extension, high-affinity binding for alpha2beta1 can control the overall platelet-adhesive activity of native collagens.

Distinct Functional Conformations of von Willebrand Factor A1 Domain in Support of Platelet-Surface or Platelet-Platelet Interactions.

The adhesive protein, von Willebrand factor (VWF), is generally considered a key substrate for platelet adhesion to the vessel wall, yet its role in platelet cohesion (aggregation) may be equally important for normal thrombus formation. In either case, the function of VWF is mediated by the primary interaction of the VWF A1 domain (VWF-A1) with glycoprotein (GP) Ibα, a component of the GPIb-IX-V receptor complex on the platelet membrane. Because normal plasma VWF in solution and GPIb coexist in circulating blood without any appreciable interaction, it has been postulated that conformational changes occur when VWF becomes immobilized and/or under the effect of pathologically elevated shear stress, such that binding to the receptor becomes possible and resultis in platelet tethering to a surface and shear-induced aggregation. Changes of the molecular shape of VWF, from coiled to extended, have been shown under the effect of hemodynamic forces, but evidence for conformational changes within VWF-A1 has remained elusive. The crystal structure of VWF-A1 in complex with a GPIbα amino terminal fragment has revealed that the VWF-A1 residues involved in the interaction are comprised between positions 544–614 and, in particular, do not include several positively charged Arg and Lys residues located in helices α4 and 5 (residues 627–668). The latter appear as likely candidates to interact with negatively charged residues in GPIbα as a consequence of potential conformational changes induced by tensile stress on the bond following an initial ligand-receptor contact. We tested this hypothesis by evaluating the ability of selected VWF-A1 mutants to support platelet adhesion or aggregation, respectively, under controlled flow conditions.Methods. We expressed in insect cells and purified a series of VWF-A1 fragments comprising residues 445–733. One fragment had native sequence and 8 had single or multiple substitutions of positively charged amino acid residues in helices α4 and/or α5. None of the substituted residues contribute to contacts with GP Ibα in the known crystal structures of the corresponding complex, and all except one were between 8 and 20 angstroms away from the closest GPIbα residue. All the fragments were dimeric (d) owing to the presence of interchain disulfide bond(s).Results: Native dVWF-A1 in solution supported platelet aggregation in a laminar flow field. Of the 8 mutants, 5 had variably decreased function (up to 95% less aggregation) and 2 had increased function (up to 200% increase in aggregation). The same results were observed with platelet-rich plasma in suspension or by measuring platelet aggregate formation with blood cells perfused over immobilized VWF-A1 at wall shear rates as high as 10,000 1/s. In contrast, as judged by the number of tethered platelets and their rolling velocities, all mutants supported adhesion as well as or better that the native VWFA-1 at all shear rates tested (500–25,000 1/s).Conclusions: These results provide structural evidence for the existence of different VWF-A1 conformers that can modulate adhesive properties with distinct effects on platelet adhesion to a surface or platelet aggregation.

In vivo thrombus formation.

Thrombus formation, including platelet adhesion, activation, secretion and aggregation as well as tissue factor-initiated thrombin generation and fibrin formation, has been studied in the past using in vitro systems, often with isolated components. Given the complexity of hemostasis and thrombosis, many of the concepts that have been developed to explain these processes are being revisited by studying thrombus formation in live animals using intravital microscopy and genetically altered mice. Although much of the dogma that has evolved has been confirmed by in vivo studies of thrombus formation, there have also been conflicts between old concepts and new direct observations. In vivo studies of the initiation of thrombus formation, platelet accumulation and thrombin generation have provided evidence for the participation of novel proteins and identified new pathways and mechanisms.

A soluble form of the Mer receptor tyrosine kinase inhibits macrophage clearance of apoptotic cells and platelet aggregation

AbstractMembrane-bound receptors generate soluble ligand-binding domains either by proteolytic cleavage of the extracellular domain or alternative mRNA splicing yielding a secreted protein. Mertk (Mer) is in a receptor tyrosine kinase family with Axl and Tyro-3, and all 3 receptors share the Gas6 ligand. Mer regulates macrophage activation, promotes apoptotic cell engulfment, and supports platelet aggregation and clot stability in vivo. We have found that the membrane-bound Mer protein is cleaved in the extracellular domain via a metalloproteinase. The cleavage results in the production of a soluble Mer protein released in a constitutive manner from cultured cells. Significant amounts of the soluble Mer protein were also detected in human plasma, suggesting its physiologic relevance. Cleavage of Mer was enhanced by treatment with LPS and PMA and was specifically inhibited by a tumor necrosis factor α-converting enzyme metalloproteinase inhibitor. As a decoy receptor for Gas6, soluble Mer prevented Gas6-mediated stimulation of membrane-bound Mer. The inhibition of Gas6 activity by soluble Mer led to defective macrophage-mediated engulfment of apoptotic cells. Furthermore, soluble Mer decreased platelet aggregation in vitro and prevented fatal collagen/epinephrine-induced thromboembolism in mice, suggesting a potential therapeutic use for soluble Mer in the treatment of clotting disorders.

Type III collagen mimetic peptides designed with anti- or pro-aggregant activities on human platelets

We report the synthesis of collagen related peptides containing the peptide sequence Lys-Hyp-Gly-Glu-Hyp-Gly-Pro-Lys. The α-triple helix peptides behave as type III collagen analogues supporting platelet aggregation, while the homotrimer which does not exhibit a triple-helical conformation inhibits type III collagen-induced human platelet aggregation. The incorporation of the octapeptide sequence in type III collagen mimetic peptides may lead to the loss of the anti-thrombotic activity for a pro-thrombotic one.