It has been known for many years that neutrophils and platelets participate in the pathogenesis of severe sepsis, but the inter-relationship between these players is completely unknown We report several cellular events that led to enhanced trapping of bacteria in blood vessels: platelet TLR4 detected TLR4 ligands in blood and induced platelet binding to adherent neutrophils This led to robust neutrophil activation and formation of neutrophil extracellular traps (NETs) Plasma from severely septic humans also induced TLR4-dependent platelet-neutrophil interactions, leading to the production of NETs The NETs retained their integrity under flow conditions and ensnared bacteria within the vasculature The entire event occurred primarily in the liver sinusoids and pulmonary capillaries, where NETs have the greatest capacity for bacterial trapping We propose that platelet TLR4 is a threshold switch for this new bacterial trapping mechanism in severe sepsis

Platelet TLR4 activates neutrophil extracellular traps to ensnare bacteria in septic blood

Background—The purpose of this statement is to provide an overview of cerebral venous sinus thrombosis and to provide recommendations for its diagnosis, management, and treatment. The intended audi...

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Diagnosis and Management of Cerebral Venous Thrombosis A Statement for Healthcare Professionals From the American Heart Association/American Stroke Association

Thrombosis of the cerebral veins and sinuses affects mainly young adults and children This potentially disabling or fatal condition is usually not diagnosed until a week after the onset of symptoms This review article describes the clinical presentations of the disorder such as those that occur after a head injury or lumbar puncture Therapeutic options include anticoagulation, thrombolysis, and measures to control intracranial hypertension

/pdf/thrombosis-of-the-cerebral-veins-and-sinuses-ducpiypk8d.pdf

Thrombosis of the cerebral veins and sinuses

Adenosine is a key endogenous molecule that regulates tissue function by activating four G-protein-coupled adenosine receptors: A1, A2A, A2B and A3. Cells of the immune system express these receptors and are responsive to the modulatory effects of adenosine in an inflammatory environment. Animal models of asthma, ischaemia, arthritis, sepsis, inflammatory bowel disease and wound healing have helped to elucidate the regulatory roles of the various adenosine receptors in dictating the development and progression of disease. This recent heightened awareness of the role of adenosine in the control of immune and inflammatory systems has generated excitement regarding the potential use of adenosine-receptor-based therapies in the treatment of infection, autoimmunity, ischaemia and degenerative diseases.

/pdf/adenosine-receptors-therapeutic-aspects-for-inflammatory-and-3bo18pvzz6.pdf

Adenosine receptors: therapeutic aspects for inflammatory and immune diseases

Antioxidant functions of carotenoids.

Background and Purpose—It has been recently reported that a G→A transition at nucleotide position 20210 in the 3′-untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increased risk of deep venous thrombosis. To date, it is unknown whether this polymorphism also represents a risk factor for cerebral venous thrombosis (CVT). Methods—Venous blood samples were collected from 45 patients with CVT and from 354 healthy blood donors as controls. A second control group consisted of 131 subjects with acute ischemic stroke or transient ischemic attack (TIA). Genomic DNA was isolated from peripheral blood leukocytes. Amplification of DNA was performed by polymerase chain reaction (PCR). The G→A transition at nucleotide position 20210 of the prothrombin gene was detected by allele-specific restriction digestion. Results—The G20210→A transition in the prothrombin gene was found in a heterozygous form in 4 of 45 patients with CVT (8.9%) and in 8 of 354 healthy control...

Prothrombin gene G20210→A transition is a risk factor for cerebral venous thrombosis

Platelet activation plays an important role in the pathomechanisms of arterial vascular disorders including stroke, peripheral arterial disease (PAD), and myocardial infarction. Circulating activated platelets may be useful markers of local thrombotic events occurring in these diseases. Using flow cytometry circulating activated platelets can be detected by determining: 1. the platelets' shape change on the basis of the different light scatter properties of discocytes and spherocytes, 2. the expression of platelet bound fibrinogen or conformation specific neoantigens on fibrinogen and on its platelet receptor, and 3. the exposure of granule membrane proteins such as P-selectin as a result of platelet secretion. The concentration-effect relationships were determined for the ADP and U46619 induced shape change, fibrinogen binding, and expression of P-selectin. The EC50 for the shape change was 4 times lower than the EC50 for the fibrinogen binding and 29 times lower than the EC50 for the expression of P-selectin. These data clearly demonstrate that the shape change is a more sensitive indicator of platelet activation in vitro than fibrinogen binding or P-selectin expression. Both the shape change and fibrinogen binding were reversible, whereas the expression of P-selectin was irreversible upon stimulation. Reversibility of the shape change may be responsible for the fact that in patients with stroke or PAD the fraction of discocytes did not differ from controls, whereas more than 80% of them revealed a significantly higher fraction of P-selectin positive platelets. Thus the determination of the P-selectin expression reveals a higher diagnostic sensitivity for detecting a platelet activation in vivo than the determination of the shape change.

Flow cytometric detection of activated platelets: comparison of determining shape change, fibrinogen binding, and P-selectin expression.

Investigations on a selective non-prostanoic thromboxane antagonist, BM 13.177, in human platelets

Determination of circulating activated platelets may be helpful to estimate the prognosis and to stratify therapies in arterial vascular disorders including stroke. We used flow cytometry and phase contrast microscopy to study whether the fraction of platelets expressing p-selectin and CD63 and the fraction of platelets with shape change are increased in patients with acute and previous cerebrovascular ischemia. The proportion of platelets expressing activation dependent antigens was higher in patients with acute (n = 24; p-selectin: 8.23 ± 4.21%; CD63: 3.53 ± 2.53%) and with previous cerebrovascular ischemia (n = 46; 3.86 ± 1.98%; 2.80 ± 1.79%) as compared to age- and sex-matched control subjects (n = 35; 2.17 ± 0.96%; 1.79 ± 0.75%; p #0.005, respectively). In patients with previous ischemia, there was no difference between treatment with aspirin (n = 25) or phenprocoumon (n = 21). Hypertension, diabetes mellitus and smoking were not associated with increased antigen expression (analysis of variance). The fraction of discoid platelets and platelet counts were not significantly different between groups. Our results indicate increased expression of platelet neoantigens in acute and to a less degree in previous cerebrovascular ischemia. Ongoing platelet activation after cerebrovascular ischemia despite therapy with aspirin or phenprocoumon indicates that new anti-platelet drugs may be of benefit for these patients. Flow cytometry appears to be a useful tool to assess platelet function in cerebrovascular ischemia.

Increased fraction of circulating activated platelets in acute and previous cerebrovascular ischemia

Mutual contacts and platelet-expressed fibrinogen seem to be required for the stimulation of neutrophils by activated platelets. The beta 2-integrins CD11b/CD18 and CD11c/CD18 are potential receptors for fibrinogen on neutrophils. In order to investigate whether binding of fibrinogen to these integrins is involved, monoclonal antibodies (MoAbs) and Gly-Pro-Arg-Pro (GPRP) peptide that inhibits fibrinogen binding to CD11c/CD18 were checked for their effects on the interaction of activated platelets and neutrophils. The luminol-amplified chemiluminescence (CL) as a measure for the oxidative burst of neutrophils was recorded simultaneously to the platelet aggregation in mixed cell suspensions. The adhesion of platelets and neutrophils was determined microscopically. The thromboxane A2 mimetic U46619 was used as a potent platelet agonist but that does not stimulate neutrophils. aggregation and a strong CL of neutrophils. The platelet-induced activation of neutrophils required added fibrinogen which fibronectin or thrombospondin could not substitute for. Cytochalasin D (Cyto D) that blocks actin polymerization totally abrogated the platelet-induced Cl of neutrophils. The MoAb OKM1 against CD11b, which blocks fibrinogen binding to CD11b/CD18 as well as the MoAbs IOT16 and IOT18 directed against CD11a and CD18, respectively, had no effect. In contrast, the MoAb LeuM5 which inhibits the binding of fibrinogen to CD11c/CD18 revealed a strong inhibition. Furthermore, GPRP peptide which CD11c/CD18 recognizes on the A alpha-chain of fibrinogen also strongly inhibited the platelet-induced CL of neutrophils, whereas control peptides such as Gly-His-Arg-Pro (GHRP) or Gly-Pro-Gly-Gly (GPGG) had no effect. In contrast to the platelet-induced CL of neutrophils, Cyto D, MoAb against CD11c and GPRP peptide did not inhibit the CL induced by FMLP and PAF in pure neutrophil suspensions. They also did not affect U46619-induced platelet aggregation. The adhesion of platelets and neutrophils was neither dependent on added fibrinogen nor inhibited by Cyto D, MoAb against CD11c and the GPRP-peptide. Therefore fibrinogen and actin polymerization seem not to be required for the adhesion of neutrophils to platelets. However, the activation of neutrophils depends on the interaction of CD11c/CD18 with the A alpha-chain of platelet-expressed fibrinogen and the contractile system of neutrophils.

Heinrich Patscheke

Papers

Prothrombin gene G20210→A transition is a risk factor for cerebral venous thrombosis

Flow cytometric detection of activated platelets: comparison of determining shape change, fibrinogen binding, and P-selectin expression.

Investigations on a selective non-prostanoic thromboxane antagonist, BM 13.177, in human platelets

Increased fraction of circulating activated platelets in acute and previous cerebrovascular ischemia

Platelet-induced neutrophil activation: platelet-expressed fibrinogen induces the oxidative burst in neutrophils by an interaction with CD11C/CD18.