CD36-fibrin interaction propagates FXI-dependent thrombin generation of human platelets.

Thrombin converts fibrinogen to fibrin and activates blood and vascular cells in thrombo-inflammatory diseases. Platelets are amplifiers of thrombin formation when activated by leukocyte- and vascular cell-derived thrombin. CD36 on platelets acts as sensitizer for molecules with damage-associated molecular patterns, thereby increasing platelet reactivity. Here, we investigated the role of CD36 in thrombin-generation on human platelets, including selected patients with advanced chronic kidney disease (CKD). Platelets deficient in CD36 or blocked by anti-CD36 antibody FA6.152 showed impaired thrombin generation triggered by thrombin in calibrated automated thrombography. Using platelets with congenital function defects, blocking antibodies, pharmacological inhibitors, and factor-depleted plasma, CD36-sensitive thrombin generation was dependent on FXI, fibrin, and platelet signaling via GPIbα and SFKs. CD36-deficiency or blocking suppressed thrombin-induced platelet αIIbß3 activation, granule exocytosis, binding of adhesion proteins and FV, FVIII, FIX, FX, but not anionic phospholipid exposure determined by flow cytometry. CD36 ligated specifically soluble fibrin, which recruited distinct coagulation factors via thiols. Selected patients with CKD showed elevated soluble fibrin plasma levels and enhanced thrombin-induced thrombin generation, which was normalized by CD36 blocking. Thus, CD36 is an important amplifier of platelet-dependent thrombin generation when exposure of anionic phospholipids is limited. This pathway might contribute to hypercoagulability in CKD.

cAMP- and cGMP-elevating agents inhibit GPIbα-mediated aggregation but not GPIbα-stimulated Syk activation in human platelets.

BACKGROUND: The glycoprotein (GP) Ib-IX-V complex is a unique platelet plasma membrane receptor, which is essential for platelet adhesion and thrombus formation. GPIbα, part of the GPIb-IX-V complex, has several physiological ligands such as von Willebrand factor (vWF), thrombospondin and distinct coagulation factors, which trigger platelet activation. Despite having an important role, intracellular GPIb-IX-V signaling and its regulation by other pathways are not well defined. Our aim was to establish the intracellular signaling response of selective GPIbα activation in human platelets, in particular the role of the tyrosine kinase Syk and its regulation by cAMP/PKA and cGMP/PKG pathways, respectively. We addressed this using echicetin beads (EB), which selectively bind to GPIbα and induce platelet aggregation. METHODS: Purified echicetin from snake Echis carinatus venom was validated by mass spectrometry. Washed human platelets were incubated with EB, in the presence or absence of echicetin monomers (EM), Src family kinase (SFK) inhibitors, Syk inhibitors and the cAMP- and cGMP-elevating agents iloprost and riociguat, respectively. Platelet aggregation was analyzed by light transmission aggregometry, protein phosphorylation by immunoblotting. Intracellular messengers inositolmonophosphate (InsP1) and Ca2+i were measured by ELISA and Fluo-3 AM/FACS, respectively. RESULTS: EB-induced platelet aggregation was dependent on integrin αIIbß3 and secondary mediators ADP and TxA2, and was antagonized by EM. EB stimulated Syk tyrosine phosphorylation at Y352, which was SFK-dependent and Syk-independent, whereas Y525/526 phosphorylation was SFK-dependent and partially Syk-dependent. Furthermore, phosphorylation of both Syk Y352 and Y525/526 was completely integrin αIIbß3-independent but, in the case of Y525/526, was partially ADP/TxA2-dependent. Syk activation, observed as Y352/ Y525/Y526 phosphorylation, led to the phosphorylation of direct substrates (LAT Y191, PLCγ2 Y759) and additional targets (Akt S473). PKA/PKG pathways inhibited EB-induced platelet aggregation and Akt phosphorylation but, surprisingly, enhanced Syk and LAT/PLCγ2 tyrosine phosphorylation. A similar PKA/PKG effect was confirmed with convulxin-/GPVI-stimulated platelets. EB-induced InsP1 accumulation/InsP3 production and Ca2+-release were Syk-dependent, but only partially inhibited by PKA/PKG pathways. CONCLUSION: EB and EM are specific agonists and antagonists, respectively, of GPIbα-mediated Syk activation leading to platelet aggregation. The cAMP/PKA and cGMP/PKG pathways do not inhibit but enhance GPIbα-/GPVI-initiated, SFK-dependent Syk activation, but strongly inhibit further downstream responses including aggregation. These data establish an important intracellular regulatory network induced by GPIbα.

High-throughput elucidation of thrombus formation reveals sources of platelet function variability.

In combination with microspotting, whole-blood microfluidics can provide high-throughput information on multiple platelet functions in thrombus formation. Based on assessment of the inter- and intra-subject variability in parameters of microspot-based thrombus formation, we aimed to determine the platelet factors contributing to this variation. Blood samples from 94 genotyped healthy subjects were analyzed for conventional platelet phenotyping: i.e. hematologic parameters, platelet glycoprotein (GP) expression levels and activation markers (24 parameters). Furthermore, platelets were activated by ADP, CRP-XL or TRAP. Parallel samples were investigated for whole-blood thrombus formation (6 microspots, providing 48 parameters of adhesion, aggregation and activation). Microspots triggered platelet activation through GP Ib-V-IX, GPVI, CLEC-2 and integrins. For most thrombus parameters, inter-subject variation was 2-4 times higher than the intra-subject variation. Principal component analyses indicated coherence between the majority of parameters for the GPVI-dependent microspots, partly linked to hematologic parameters, and glycoprotein expression levels. Prediction models identified parameters per microspot that were linked to variation in agonist-induced αIIbß3 activation and secretion. Common sequence variation of GP6 and FCER1G, associated with GPVI-induced αIIbß3 activation and secretion, affected parameters of GPVI-and CLEC-2-dependent thrombus formation. Subsequent analysis of blood samples from patients with Glanzmann thrombasthenia or storage pool disease revealed thrombus signatures of aggregation-dependent parameters that were subject-dependent, but not linked to GPVI activity. Taken together, this high-throughput elucidation of thrombus formation revealed patterns of inter-subject differences in platelet function, which were partly related to GPVI-induced activation and common genetic variance linked to GPVI, but also included a distinct platelet aggregation component.

Diagnosis and Management of Inherited Platelet Disorders.

In clinical daily practice the definition of a bleeding tendency is rather subjective. Clinical manifestations usually include hematoma, epistaxis, menorrhagia, and severe bleeding episodes after surgery or injuries. The most common causes are disorders of primary hemostasis that occur sometimes due to platelet function disorders. Inherited thrombocytopathies are much less frequent in comparison to acquired platelet function disorders. However, congenital disorders can lead to severe bleeding tendency and are often not diagnosed. They are induced by different platelet defects based on disorders of platelet adhesion, receptors, secretion, and signal transduction. In some cases, they are associated with thrombocytopenias, giant platelets, and various comorbidities. This article gives an overview of the different defects, their diagnosis, and treatment options.

Novel Mutations in the GPIIb and GPIIIa Genes in Glanzmann Thrombasthenia.

BACKGROUND: Glanzmann thrombasthenia (GT) is an inherited autosomal recessive platelet disorder characterized by a complete or partial lack, or mutation, of the GPIIb/IIIa complex (integrin α(IIb)ß(3)) on the thrombocytes' surface, leading to a severe bleeding syndrome. MATERIAL AND METHODS: Molecular genetic analysis was performed in patients with suspected GT. The aim of the present study was the identification of new natural variants, their impact on platelet function, and their relation to the risk of bleeding. RESULTS: Expression of the platelet integrin α(IIb)ß(3) was determined by flow cytometry. Mutations were identified through sequencing of cDNA and genomic DNA. In addition, platelet function studies (PAC-binding, aggregations) were implemented. The study included 25 patients revealing 13 mutations (GPIIb: n = 9; GPIIIa: n = 4). Two of the 13 mutations were previously described (T207I; L214P). The remaining mutations have not been published yet, whereas 1 mutation in 2 unrelated families was identical (3062 T→C). CONCLUSION: All patients with less than 25% of present α(IIb)ß(3) have a medical history of bleeding.

Thrombin-induced interleukin 1beta synthesis in platelet suspensions: impact of contaminating leukocytes.

A controversial discussion as to whether human platelets are capable of regulated protein synthesis has been ongoing for over half a century. A previous study has suggested that human platelets synthesize large amounts of interleukin 1beta (IL-1beta) in response to external cues and in a physiologically significant manner. However, cytokines such as IL-1beta are generally considered to be products of leukocytes and it could not be completely excluded that contaminating leukocytes may have contributed to the IL-1beta results in platelet preparations. It was therefore our intention to investigate whether residual leukocytes had an impact on thrombin-induced IL-1beta synthesis. Using various methods to reduce the level of contaminating leukocytes, we found that IL-1beta production in platelet-rich suspensions is dependent on the presence of leukocytes, as it was decreased by reducing the number of leukocytes. In addition, we found that thrombin-induced IL-1beta synthesis was completely eliminated in leukocyte-free platelet preparations and could be restored by adding leukocytes. IL-1beta synthesis could be detected in platelet suspensions contaminated with at least 1 leukocyte per 10(5) platelets. This study demonstrated that platelets are incapable of synthesizing detectable amounts of IL-1beta on their own. We suggest that any IL-1beta synthesis detected is a by-product of leukocytes contaminating the platelet preparations. Thus, the hypothesis that platelets producing IL-1beta, provide a new link between thrombosis and inflammation needs to be reconsidered.

Multicenter study on the diagnostic value of a new RIA for the detection of free plasma metanephrines in the work-up for pheochromocytoma.

Available laboratory test methods for the detection of elevated concentrations of catecholamines and their metabolites in urine and/or plasma are not always sensitive enough for the detection of pheochromocytoma. High-quality immunoassays for these compounds appear to be as accurate as high-pressure liquid chromatography (HPLC) or gas chromatography/mass spectrometry (GC-MS). Therefore, the current project aims to establish a new sensitive radioimmunoassay (RIA) for the measurement of free metanephrines in the plasma of patients in the work-up for pheochromocytoma. We report first results of an ongoing multicenter clinico-chemical evaluation study in hypertensive patients and normotensive volunteers. After an overnight fast plasma samples were collected on ice in EDTA- and heparin-coated tubes after insertion of an indwelling venous line and resting in the supine (patients) or sitting position (normal volunteers) for 30 min. Plasma metanephrines were measured by a newly developed RIA from IBL, Hamburg, Germany. Good agreement of the assay with the tandem mass spectrometry (LC-MS/MS) method for normetanephrine (r2=0.975) and for metanephrine (r2=0.985) could be demonstrated. Both specimens, EDTA and heparin plasma, can be used with the same results. The RIA has a good precision of <15% in the normal range and of <10% in the elevated concentration range. Our preliminary data suggest a high validity of the newly developed RIA for measuring free metanephrine and normetanephrine in hypertensive subjects in both EDTA and heparin plasma. Further work is required to determine the accuracy of the test in larger patient populations and in patients with pheochromocytoma.