Leukocyte adhesion deficiency-III (LAD-III) also called leukocyte adhesion deficiency-1/variant (LAD1v) is a rare congenital disease caused by defective integrin activation of leukocytes and ...platelets. Patients with LAD-III present with non-purulent infections and increased bleeding symptoms. We report on a novel integrin-dependent platelet dysfunction in two brothers with LAD-III syndrome caused by a homozygous mutation 1717C>T in the FERMT3 gene leading to a premature stop codon R573X in the focal adhesion protein kindlin-3. Stimulation of patients´ platelets with all used agonists resulted in a severely decreased binding of soluble fibrinogen indicating a defect in inside-out activation of the integrin α IIb β 3 (GPIIb/IIIa). Patients´ platelets did not respond to the α 2 β 1 -integrin agonist aggretin-A at all. Our data on granula secretion indicate for the first time that the thrombin receptor PAR-4 but not PAR-1 may be important in integrin-triggered granule secretion in response to thrombin. In contrast, collagen mediated platelet granule secretion was not affected in LAD-III-patients. Thus, integrin-signalling may be not essential in collagen-induced granule secretion. The patients’ peripheral blood mononuclear cells showed a severe loss of adhesion capacity to VCAM-1 and to endothelial cells compared to cells from healthy donors. Rap-1 activation after PMA stimulation could be observed in controls´ but not in patients´ cells. After haematogenesis stem cell transplantation (HSCT) the brothers showed no symptoms of bleeding or immunodeficiency and the integrin-dependent platelet and leukocyte functions normalised.
Summary
Platelet activation is involved in the pathogenesis of cerebrovascular ischemia, but the major agonist involved has yet to be identified. To investigate the role of thrombin in platelet ...activation in patients with acute ischemic stroke, and while thrombin is the most likely candidate for activation of the thrombin receptor PAR-1
in vivo
, we assessed its cleavage and internalization using the antibodies SPAN12, binding to uncleaved PAR-1, and WEDE15, recognizing cleaved and uncleaved, but not internalized PAR-1. In contrast to healthy age-matched controls, platelets from stroke patients exhibited significant cleavage and internalization of PAR-1 (P<0.001) and failed to respond to thrombin
in vitro
. Enhanced surface expression of CD62P, CD63, TSP-1 and less mepacrine uptake showed platelet degranulation during stroke. Platelets from patients with acute cerebral ischemia are exhausted and desensitized to thrombin through cleavage of PAR-1, indicating that high concentrations of thrombin occur with acute cerebrovascular ischemic events
in vivo
.
Convulxin, a powerful platelet activator, was isolated from Crotalus durissus terrificus venom, and 20 amino acid N-terminal sequences of both subunits were determined. These indicated that convulxin ...belongs to
the heterodimeric C-type lectin family. Neither antibodies against GPIb nor echicetin had any effect on convulxin-induced
platelet aggregation showing that, in contrast to other venom C-type lectins acting on platelets, GPIb is not involved in
convulxin-induced platelet activation. In addition, partially reduced/denatured convulxin only affects collagen-induced platelet
aggregation. The mechanism of convulxin-induced platelet activation was examined by platelet aggregation, detection of time-dependent
tyrosine phosphorylation of platelet proteins, and binding studies with 125 I-convulxin. Convulxin induces signal transduction in part like collagen, involving the time-dependent tyrosine phosphorylation
of Fc receptor γ chain, phospholipase Cγ2, p72 SYK , c-Cbl, and p36â38. However, unlike collagen, pp125 FAK and some other bands are not tyrosine-phosphorylated. Convulxin binds to a glycosylated 62-kDa membrane component in platelet
lysate and to p62/GPVI immunoprecipitated by human anti-p62/GPVI antibodies. Convulxin subunits inhibit both aggregation and
tyrosine phosphorylation in response to collagen. Piceatannol, a tyrosine kinase inhibitor with some specificity for p72 SYK , showed differential effects on collagen and convulxin-stimulated signaling. These results suggest that convulxin uses the
p62/GPVI but not the α 2 β 1 part of the collagen signaling pathways to activate platelets. Occupation and clustering of p62/GPVI may activate Src family
kinases phosphorylating Fc receptor γ chain and, by a mechanism previously described in T- and B-cells, activate p72 SYK that is critical for downstream activation of platelets.
The snake venom C-type lectin alboaggregin A (or 50-kd alboaggregin) from Trimeresurus albolabris was previously shown to be a platelet glycoprotein (GP) Ib agonist. However, investigations of the ...signal transduction induced in platelets showed patterns of tyrosine phosphorylation that were different from those of other GPIb agonists and suggested the presence of an additional receptor. In this study, the binding of biotinylated alboaggregin A to platelet lysates, as well as affinity chromatography evaluations of platelet lysates on an alboaggregin A-coated column, indicated that this other receptor is GPVI. Additional experiments with reagents that inhibit either GPIb or GPVI specifically supported this finding. These experiments also showed that both GPIb and GPVI have a role in the combined signaling and that the overall direction this takes can be influenced by inhibitors of one or the other receptor pathway.
Thrombospondin-1 (TSP1) is a matricellular glycoprotein that has key roles in interactions between human cells and components of the extracellular matrix. Here we report a novel role for the lectin ...TSP1 in pathogen-host interactions. Binding assays and flow cytometric analysis demonstrate that Streptococcus pneumoniae and other Gram-positive pathogens including S. pyogenes, Staphylococcus aureus, and Listeria monocytogenes interact specifically with human TSP1. We also show for the first time that host cell-bound TSP1 promotes adherence of Gram-positive pathogens to human epithelial and endothelial cell lines. Pretreatment of bacteria with sodium periodate but not Pronase E substantially reduced TSP1-mediated bacterial adherence to host cells, suggesting that a glycoconjugate on the bacterial cell surface functions as the receptor for TSP1. Lipoteichoic acids did not affect TSP1-mediated adherence of S. pneumoniae to host cells. In contrast, attachment of S. pneumoniae and other Gram-positive pathogens to host cells via TSP1 was blocked by soluble peptidoglycan, indicating recognition of bacterial peptidoglycan by TSP1. In conclusion, our results demonstrate that recognition of Gram-positive pathogens by TSP1 promotes bacterial colonization of host tissue cells. In this scenario, peptidoglycan functions as adhesin and TSP1 acts as a molecular bridge linking Gram-positive bacteria with receptors on the host cell.--Rennemeier, C., Hammerschmidt, S., Niemann, S., Inamura, S., Zähringer, U., Kehrel, B. E. Thrombospondin-1 promotes cellular adherence of Gram-positive pathogens via recognition of peptidoglycan.
Platelets bind to Candida albicans, the major cause of candidiasis. But in contrast to other microorganisms the fungus does not aggregate platelets. Gliotoxin (GT), which possesses immunosuppressive ...properties, is produced by various fungi, including the opportunistic pathogens Aspergillus fumigatus and C. albicans . Its mode of action involves the formation of mixed disulfides with host proteins. Disulfide exchanges play an important role in platelet activation. Therefore, the effect of C. albicans and GT on platelet function was tested. C. albicans yeast cells (5,000–10,000 cells/μl) and GT, in pathophysiologically relevant concentrations (0.05–0.5 μM), inhibited platelet fibrinogen binding, anti gp IIb/IIIa antibody PAC-1 binding, aggregation and procoagulant activity in a dose-dependent manner. Alpha granule release, measured via CD62P surface expression, was not affected. Addition of reduced glutathione partially counteracted the effect of C. albicans and GT on platelet fibrinogen binding and platelet aggregation. The C. albicans metabolite GT features antithrombotic properties in addition to its immunosuppressive functions. Since treatment with reduced glutathione partially counteracted the inhibitory effect of C. albicans yeast cells and GT on platelet fibrinogen binding, the antithrombotic activity is likely to depend on the disulfide bridge of this mycotoxin. GT production by C. albicans could contribute to its survival in the blood stream during vascular infections. The knowledge of the underlying mechanisms of the antithrombotic properties might help to treat fungal infections as well as thrombosis.
The adherence of microorganisms to platelets previously immobilized on the subendocardium in nonbacterial thrombotic endocarditis is considered an important pathogenic step in Staphylococcus aureus ...endocarditis. To identify and characterize bacterial factors involved in the adherence to platelets, a phage display library of S. aureus was generated by use of the phagemid pG8H6. The library was affinity panned against purified immobilized platelets. After a second panning against platelets, a significant increase in the number of eluted phagemid particles was observed; 27% of 88 randomly isolated clones expressed overlapping deduced amino acid sequences with high similarity to the C-terminal domain of the S. aureus coagulase. In addition, 22% of the clones expressed the N-terminal domain of the fibrinogen-binding protein Efb. The surface-associated fraction of the C-terminal domain of coagulase or the N-terminal domain of Efb may be involved in bacterial adherence to immobilized platelets, and fibrinogen may act as a bridging molecule in that interaction
Studies have suggested a pivotal role for free sulfhydryls in platelet integrin function, and enzyme-mediated reduction of disulfide bonds on platelets has been implicated. The platelet fibrinogen ...receptor αIIbβ3is the best-studied platelet integrin and serves as a model system for studying the structure-function relation in this family of adhesion receptors. The demonstration of free sulfhydryls on the exofacial domain of purified αIIbβ3, specifically in its activated conformation, prompted us to explore the potential for activation-dependent, enzymatically catalyzed thiol expression on intact platelets and the possible role of surface-associated protein disulfide isomerase (PDI) in αIIbβ3ligation. Using the membrane-impermeant sulfhydryl blocker para-chloromercuriphenyl sulfonate, the inhibitor of disulfide exchange bacitracin, and the monoclonal anti-PDI antibody RL90, we examined fibrinogen binding to αIIbβ3as well as ligation-induced allosteric changes in the conformation of αIIbβ3. We sought to distinguish the possible involvement of disulfide exchange in agonist-induced platelet stimulation from its role in integrin ligation. Analysis of the role of free thiols in platelet aggregation suggested a thiol-independent initial ligation followed by a thiol-dependent stabilization of binding. Flow cytometric analysis showed that sustained binding of fibrinogen, as well as expression of ligand-induced binding site epitopes and ligand-bound conformation, depended on free thiols and disulfide exchange. Expression of P-selectin was minimally affected, even with complete inhibition of αIIbβ3function. These data indicate that although agonist-induced platelet stimulation is independent of ecto-sulfhydryls, engagement of integrin αIIbβ3on the intact platelet depends totally on their enzymatically catalyzed surface expression.
ABSTRACT
Acute thrombotic arterial occlusion is the leading cause of morbidity and mortality in the Western world. Von Willebrand factor is thought to be the only indispensable adhesive substrate to ...promote thrombus formation in high shear environments. We found that thrombospondin‐1, a glycoprotein enriched in arteriosclerotic plaques, might function as an alternative substrate for thrombus formation. Platelets adhered to thrombospondin‐1 in a shear dependent manner with an optimum shear as found in stenosed arteries. Adhesion is extremely firm, with no detachment of platelets up to a shear rate of 4000 s−1. Experiments using platelets from a patient completely lacking von Willebrand factor showed that von Willebrand factor is not involved in platelet binding to thrombospondin‐1. Platelet adhesion to thrombospondin‐1 is not mediated via β3‐integrins or GPIa. CD36 partially mediates the adhesion of pre‐activated platelets. We identified GPIb as high shear adhesion‐receptor for thrombospondin‐1. Soluble GPIb, as well as antibodies against the GPIb, blocked platelet adhesion almost completely. The new discovered thrombospondin‐1‐GPIb adhesion axis under arterial shear conditions might be important, not only during thrombus formation but also for pathological processes where other cells bind to the endothelium or subendothelium, including arteriosclerosis, inflammation and tumor metastasis, and a promising therapeutic target.
Arginine vasopressin (AVP) is increasingly used in the therapy of septic patients with hypotension. However, its effects on the microvascular networks have not been studied in detail. This study was ...designed to determine the effects of AVP infusion on the villus microcirculation of the septic rat ileum.
Prospective, placebo-controlled, randomized, single-blinded trial.
University research laboratory.
Fifteen male Sprague-Dawley rats.
Twenty-four hours after cecal ligation and perforation to create sepsis (M1), rats (n = 8) received a continuous AVP infusion to increase mean arterial pressure by 20 mm Hg (M2) and 40 mm Hg (M3) from M1. In the control group (n = 7), an equivalent volume of normal saline was infused.
Videomicroscopy was performed on 6-10 villi of ileum mucosa at M1 and was repeated at M2 and M3. Blood was drawn to determine plasma levels of AVP and interleukin-6. At M1, both study groups were hypotensive compared with preseptic data (mean arterial pressure, -25%). The increase in mean arterial pressure was linked to supraphysiologic AVP plasma levels and was accompanied by a decrease in mean mucosal blood flow by 76% at M2 and 81% at M3 (p <.001 vs. control). Red blood cell velocity fell by 45% and 47%, respectively (p <.05 vs. control). Whereas periods of arrested villus blood flow increased from 8.1 +/- 2.6 secs/min to 43.8 +/- 5.2 and 47 +/- 6.2 secs/min at M2 and M3 (p <.001), the diameter of terminal arterioles remained unchanged. In addition, AVP infusion further augmented the sepsis-associated increase in interleukin-6 levels (AVP, 905 +/- 160 vs. control, 638 +/- 55 pg/mL; p =.022).
This study provides evidence for severe abnormalities in gut mucosal blood flow after AVP infusion in septic rats, accompanied by an augmented inflammatory response to the septic injury. The effects of AVP on microvascular blood flow in this model may be related to AVP activities on larger arterioles (>40 microm), a concomitant reduction in cardiac output, or even both.
PDF
