NET burden in left atrial blood is associated with biomarkers of thrombosis and cardiac injury in patients with enlarged left atria.

BACKGROUND: Emerging data suggest an association between left atrial (LA) enlargement, thrombus formation, and ischemic stroke. However, it is unknown what may mediate such clot formation in LA dysfunction. Neutrophils promote large vessel occlusion and microthrombosis via neutrophil extracellular trap (NET) release, thus lying at the interface of inflammation, thrombosis, and fibrosis. APPROACH: We conducted a prospective all-comers cohort study in patients undergoing catheterization procedures with atrial transseptal access (MitraClip, MC; left atrial appendage closure, LAAC; pulmonary vein ablation, PVA; patent foramen ovale closure, PFO). We measured NETs, cytokines, thrombotic factors, and cardiac injury markers in paired blood samples collected from peripheral blood and within the left atrium. We correlated these biomarkers with echocardiographic measures of LA structure and function (including left atrial volume index, LAVI). Data were analyzed by procedure type, and stratified by LAVI or atrial fibrillation (AF) status. RESULTS: We enrolled 70 patients (mean age 64 years, 53% women). NETs, but not other markers, were elevated in LA compared to peripheral blood samples. Most thrombotic, inflammatory, and cardiac damage markers were elevated in LAs from MC or LAAC compared to PFO patients. Overall, NET biomarkers positively correlated with VWF, LAVI, and markers of cardiac injury and negatively with ADAMTS13 activity. LA enlargement and the presence of AF similarly stratified patients based on thromboinflammation measurements, but this was not limited to AF at the time of sample collection. CONCLUSION: Elevated NETs and VWF in patients with enlarged LA or AF suggest enhanced thromboinflammation within the LA.

Decreased Platelet Aggregation in Patients with Decompensated Liver Cirrhosis and TIPS Implantation.

Transjugular intrahepatic portosystemic shunt (TIPS) implantation is an effective treatment of portal hypertension in patients with decompensated liver cirrhosis. However, some patients develop TIPS thrombosis with recurrence of portal hypertension. The role of platelets in TIPS thrombosis and the necessity of antiplatelet therapy is unclear. Therefore, we aimed to study platelet function in patients with liver cirrhosis prior to and after TIPS implantation. Platelet aggregation was tested in peripheral and portal-vein blood patient samples on the day (D) of TIPS implantation (D0), D4 and D30 following the procedure (platelet count above 100 × 103/µL, aspirin starting on D5) using whole-blood impedance aggregometry (WBIA) and light transmission aggregometry (LTA). In addition, surface platelet activation markers (P-selectin, activated GPIIb/IIIa) and platelet-neutrophil complexes (PNCs) were assessed by flow cytometry. Thrombin receptor activating peptide 6 (TRAP-6), adenosine diphosphate (ADP) and arachidonic acid (AA) were used as agonists. Healthy subjects were included as controls. Agonist-induced platelet aggregation was reduced (WBIA: TRAP-6 p < 0.01, ADP p < 0.01, AA p < 0.001; LTA: TRAP-6 p = 0.13, ADP p = 0.05, AA p < 0.01) in patients (D0, n = 13) compared with healthy subjects (n = 9). While surface activation markers at baseline were negligibly low, the percentage of PNCs was higher in patients than in controls (p < 0.05). ADP-induced P-selectin expression was increased (p < 0.001), whereas TRAP-6-induced GPIIb/IIIa activation was impaired (p < 0.001) in patients versus controls. PNC formation in response to agonists was not different between groups. Results did not differ between peripheral and portal-vein blood of patients (D0, n = 11) and did not change over time (D0, D4, D30) following TIPS implantation (n = 9). In summary, patients with decompensated liver cirrhosis display in vitro platelet aggregation defects in response to various agonists. Defective aggregation persists upon TIPS implantation. Therefore, we conclude that antiplatelet treatment to prevent TIPS thrombosis is questionable.

Serotonin transporter-deficient mice display enhanced adipose tissue inflammation after chronic high-fat diet feeding.

Introduction: Serotonin is involved in leukocyte recruitment during inflammation. Deficiency of the serotonin transporter (SERT) is associated with metabolic changes in humans and mice. A possible link and interaction between the inflammatory effects of serotonin and metabolic derangements in SERT-deficient mice has not been investigated so far. Methods: SERT-deficient (Sert -/-) and wild type (WT) mice were fed a high-fat diet, starting at 8 weeks of age. Metabolic phenotyping (metabolic caging, glucose and insulin tolerance testing, body and organ weight measurements, qPCR, histology) and assessment of adipose tissue inflammation (flow cytometry, histology, qPCR) were carried out at the end of the 19-week high-fat diet feeding period. In parallel, Sert -/- and WT mice received a control diet and were analyzed either at the time point equivalent to high-fat diet feeding or as early as 8-11 weeks of age for baseline characterization. Results: After 19 weeks of high-fat diet, Sert -/- and WT mice displayed similar whole-body and fat pad weights despite increased relative weight gain due to lower starting body weight in Sert -/-. In obese Sert -/- animals insulin resistance and liver steatosis were enhanced as compared to WT animals. Leukocyte accumulation and mRNA expression of cytokine signaling mediators were increased in epididymal adipose tissue of obese Sert -/- mice. These effects were associated with higher adipose tissue mRNA expression of the chemokine monocyte chemoattractant protein 1 and presence of monocytosis in blood with an increased proportion of pro-inflammatory Ly6C+ monocytes. By contrast, Sert -/- mice fed a control diet did not display adipose tissue inflammation. Discussion: Our observations suggest that SERT deficiency in mice is associated with inflammatory processes that manifest as increased adipose tissue inflammation upon chronic high-fat diet feeding due to enhanced leukocyte recruitment.

Early SARS-CoV-2 infection: Platelet-neutrophil complexes and platelet function.

Background: Conflicting results have been reported on platelet activity ex vivo and responsiveness in vitro among patients with COVID-19 with or without thromboembolic complications. Objectives: To assess platelet reactivity in patients with moderate disease at early stages of COVID-19. Methods: We performed a prospective, descriptive analysis of 100 consecutive patients presenting with suspected SARS-CoV-2 infection at University Medical Center Freiburg during the first or second wave of the pandemic. Following polymerase chain reaction testing and compliance with study inclusion criteria, 20 SARS-CoV-2-positive and 55 SARS-CoV-2-negative patients (serving as patient controls) were enrolled. In addition, 15 healthy subjects were included. Platelet reactivity was assessed using whole-blood impedance aggregometry and flow cytometry in response to various agonists. Results: Platelet aggregation was significantly impaired in the patients with COVID-19 compared with that in the patient controls or healthy subjects. The reduced platelet responsiveness in the patients with COVID-19 was associated with impaired activation of GPIIb/IIIa (αIIbß3). In contrast, low expression of P-selectin at baseline and intact secretion upon stimulation in vitro suggest that no preactivation in vivo, leading to "exhausted" platelets, had occurred. The proportion of circulating platelet-neutrophil complexes was significantly higher in the patients with COVID-19 (mean ± SD, 41% ± 13%) than in the patient controls (18% ± 7%; 95% CI, 11.1-34.1; P = .0002) or healthy subjects (17% ± 4%; 95% CI, 13.8-33.8; P < .0001). An analysis of neutrophil adhesion receptors revealed upregulation of CD11b (α-subunit of αMß2) and CD66b (CEACAM8) but not of CD162 (PSGL-1) in the patients with COVID-19. Conclusion: Despite reduced platelet responsiveness, platelet-neutrophil complexes are increased at early stages of moderate disease. Thus, this cellular interaction may occur during COVID-19 without preceding platelet activation.

Platelet Subtypes in Inflammatory Settings.

In addition to their essential role in hemostasis and thrombosis, platelets also modulate inflammatory reactions and immune responses. This is achieved by specialized surface receptors as well as secretory products including inflammatory mediators and cytokines. Platelets can support and facilitate the recruitment of leukocytes into inflamed tissue. The various properties of platelet function make it less surprising that circulating platelets are different within one individual. Platelets have different physical properties leading to distinct subtypes of platelets based either on their function (procoagulant, aggregatory, secretory) or their age (reticulated/immature, non-reticulated/mature). To understand the significance of platelet phenotypic variation, qualitatively distinguishable platelet phenotypes should be studied in a variety of physiological and pathological circumstances. The advancement in proteomics instrumentation and tools (such as mass spectrometry-driven approaches) improved the ability to perform studies beyond that of foundational work. Despite the wealth of knowledge around molecular processes in platelets, knowledge gaps in understanding platelet phenotypes in health and disease exist. In this review, we report an overview of the role of platelet subpopulations in inflammation and a selection of tools for investigating the role of platelet subpopulations in inflammation.

Platelet olfactory receptor activation limits platelet reactivity and growth of aortic aneurysms.

As blood transitions from steady laminar flow (S-flow) in healthy arteries to disturbed flow (D-flow) in aneurysmal arteries, platelets are subjected to external forces. Biomechanical platelet activation is incompletely understood and is a potential mechanism behind antiplatelet medication resistance. Although it has been demonstrated that antiplatelet drugs suppress the growth of abdominal aortic aneurysms (AAA) in patients, we found that a certain degree of platelet reactivity persisted in spite of aspirin therapy, urging us to consider additional antiplatelet therapeutic targets. Transcriptomic profiling of platelets from patients with AAA revealed upregulation of a signal transduction pathway common to olfactory receptors, and this was explored as a mediator of AAA progression. Healthy platelets subjected to D-flow ex vivo, platelets from patients with AAA, and platelets in murine models of AAA demonstrated increased membrane olfactory receptor 2L13 (OR2L13) expression. A drug screen identified a molecule activating platelet OR2L13, which limited both biochemical and biomechanical platelet activation as well as AAA growth. This observation was further supported by selective deletion of the OR2L13 ortholog in a murine model of AAA that accelerated aortic aneurysm growth and rupture. These studies revealed that olfactory receptors regulate platelet activation in AAA and aneurysmal progression through platelet-derived mediators of aortic remodeling.

ICODE: the international COVID-19 thrombosis biomarkers colloquium-novel soluble biomarkers: circulating cell-free nucleic acids and other molecules.

This article summarizes the evidence derived from clinical (observational) studies describing novel soluble biomarkers in COVID-19. Our goal was to stimulate further research (preclinical as well as clinical studies) and therefore we discuss potential prognostic value, but also technical details, such as sample preparation. A table provides an overview of the described biomarkers measured in plasma, serum or other (namely bronchoalveolar) fluids.

Complement system component dysregulation is a distinctive feature of COVID-19 disease: a prospective and comparative analysis of patients admitted to the emergency department for suspected COVID-19 disease.

The complement system (CS) plays a pivotal role in Coronavirus disease 2019 (COVID-19) pathophysiology. The objective of this study was to provide a comparative, prospective data analysis of CS components in an all-comers cohort and COVID-19 patients. Patients with suspected COVID-19 infection admitted to the Emergency department were grouped for definite diagnosis of COVID-19 and no COVID-19 accordingly. Clinical presentation, routine laboratory and von Willebrand factor (vWF) antigen as well as CS components 3, 4 and activated 5 (C5a) were assessed. Also, total complement activity via the classical pathway (CH50) was determined. Levels of calprotectin in serum were measured using an automated quantitative lateral flow assay. We included 80 patients in this prospective trial. Of those 19 (23.7%) were tested positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Patients with COVID-19 had higher levels of CS components 5a and 4 (54.79 [24.14-88.79] ng/ml vs. 35 [23.15-46.1] ng/ml; p = 0.0433 and 0.3772 [± 0.1056] g/L vs. 0.286 [0.2375-0.3748] g/L; p = 0.0168). COVID-19 patients had significantly higher levels of vWF antigen when compared to the control group (288.3 [± 80.26] % vs. 212 [151-320] %; p = 0.0469). There was a significant correlation between CS C3 and 5a with vWF antigen (rs = 0.5957 [p = 0.0131] and rs = 0.5015 [p = 0.042]) in COVID-19 patients. There was no difference in calprotectin plasma levels (4.786 [± 2.397] µg/ml vs. 4.233 [± 2.142] µg/ml; p = 0.4175) between both groups. This prospective data from a single centre all-comers cohort accentuates altered levels of CS components as a distinct feature of COVID-19 disease. Deregulation of CS component 3 and C5a are associated with increased vWF antigen possibly linking vascular damage to alternative CS activation in COVID-19.

Peripheral serotonin lacks effects on endothelial adhesion molecule expression in acute inflammation.

BACKGROUND: Peripheral, non-neuronal serotonin promotes the recruitment of neutrophils to sites of acute inflammation and tissue damage. Direct effects of serotonin on neutrophil function were shown to be involved. However, the influence of serotonin on the endothelial counterpart is unknown. OBJECTIVES: To investigate whether serotonin alters the function of endothelial cells in leukocyte recruitment during acute inflammation. METHODS: We used two murine models of acute inflammation: intraperitoneal lipopolysaccharide (LPS) injection and mesenteric ischemia/reperfusion injury (I/R). To study effects of peripheral serotonin, leukocyte recruitment and endothelial adhesion molecule expression were compared in wild type (WT) and tryptophan hydroxylase 1 deficient (Tph1-/- ) mice, which are unable to synthesize peripheral serotonin. RESULTS: As expected, neutrophil transmigration into the peritoneal cavity following LPS injection was impaired in Tph1-/- mice. Abdominal blood vessels, however, showed no difference in adhesion molecule expression. In the early reperfusion phase after mesenteric I/R, the number of rolling leukocytes was significantly lower in Tph1-/- compared to WT. In line with the LPS model, endothelial adhesion molecule expression was independent of serotonin. In vitro experiments using human umbilical vein endothelial cells (HUVECs) confirmed that serotonin does not affect endothelial adhesion molecules. CONCLUSIONS: The inflammatory release of peripheral serotonin is dispensable for the regulation of endothelial adhesion molecules.

Thromboinflammation as a Driver of Venous Thromboembolism.

Thrombus formation has been identified as an integral part in innate immunity, termed immunothrombosis. Activation of host defense systems is known to result in a procoagulant environment. In this system, cellular players as well as soluble mediators interact with each other and their dysregulation can lead to the pathological process of thromboinflammation. These mechanisms have been under intensified investigation during the COVID-19 pandemic. In this review, we focus on the underlying mechanisms leading to thromboinflammation as one trigger of venous thromboembolism.

Platelet MHC class I mediates CD8+ T-cell suppression during sepsis.

Circulating platelets interact with leukocytes to modulate host immune and thrombotic responses. In sepsis, platelet-leukocyte interactions are increased and have been associated with adverse clinical events, including increased platelet-T-cell interactions. Sepsis is associated with reduced CD8+ T-cell numbers and functional responses, but whether platelets regulate CD8+ T-cell responses during sepsis remains unknown. In our current study, we systemically evaluated platelet antigen internalization and presentation through major histocompatibility complex class I (MHC-I) and their effects on antigen-specific CD8+ T cells in sepsis in vivo and ex vivo. We discovered that both human and murine platelets internalize and proteolyze exogenous antigens, generating peptides that are loaded onto MHC-I. The expression of platelet MHC-I, but not platelet MHC-II, is significantly increased in human and murine platelets during sepsis and in human megakaryocytes stimulated with agonists generated systemically during sepsis (eg, interferon-γ and lipopolysaccharide). Upregulation of platelet MHC-I during sepsis increases antigen cross-presentation and interactions with CD8+ T cells in an antigen-specific manner. Using a platelet lineage-specific MHC-I-deficient mouse strain (B2Mf/f-Pf4Cre), we demonstrate that platelet MHC-I regulates antigen-specific CD8+ T-cell proliferation in vitro, as well as the number and functional responses of CD8+ T cells in vivo, during sepsis. Loss of platelet MHC-I reduces sepsis-associated mortality in mice in an antigen-specific setting. These data identify a new mechanism by which platelets, through MHC-I, process and cross-present antigens, engage antigen-specific CD8+ T cells, and regulate CD8+ T-cell numbers, functional responses, and outcomes during sepsis.

Heparanase expression and activity are increased in platelets during clinical sepsis.

BACKGROUND: Heparanase (HPSE) is the only known mammalian enzyme that can degrade heparan sulfate. Heparan sulfate proteoglycans are essential components of the glycocalyx, and maintain physiological barriers between the blood and endothelial cells. HPSE increases during sepsis, which contributes to injurious glyocalyx degradation, loss of endothelial barrier function, and mortality. OBJECTIVES: As platelets are one of the most abundant cellular sources of HPSE, we sought to determine whether HPSE expression and activity increases in human platelets during clinical sepsis. We also examined associations between platelet HPSE expression and clinical outcomes. PATIENTS/METHODS: Expression and activity of HPSE was determined in platelets isolated from septic patients (n = 59) and, for comparison, sex-matched healthy donors (n = 46) using complementary transcriptomic, proteomic, and functional enzymatic assays. Septic patients were followed for the primary outcome of mortality, and clinical data were captured prospectively for septic patients. RESULTS: The mRNA expression of HPSE was significantly increased in platelets isolated from septic patients. Ribosomal footprint profiling, followed by [S35] methionine labeling assays, demonstrated that HPSE mRNA translation and HPSE protein synthesis were significantly upregulated in platelets during sepsis. While both the pro- and active forms of HPSE protein increased in platelets during sepsis, only the active form of HPSE protein significantly correlated with sepsis-associated mortality. Consistent with transcriptomic and proteomic upregulation, HPSE enzymatic activity was also increased in platelets during sepsis. CONCLUSIONS: During clinical sepsis HPSE, translation, and enzymatic activity are increased in platelets. Increased expression of the active form of HPSE protein is associated with sepsis-associated mortality.

Sepsis alters the transcriptional and translational landscape of human and murine platelets.

There is increasing recognition that platelets have a functional role in the pathophysiology of sepsis, though this role has not been precisely defined. Whether sepsis alters the human platelet transcriptome and translational landscape has never been established. We used parallel techniques of RNA sequencing and ribosome footprint profiling to interrogate the platelet transcriptome and translatome in septic patients and healthy donors. We identified 1806 significantly differentially expressed (false discovery rate <0.05) transcripts in platelets from septic patients. Platelet translational events during sepsis were also upregulated. To explore the relevance of a murine model of sepsis, cecal ligation and puncture (CLP), we compared sepsis-induced changes in platelet gene expression between septic patients and mice subjected to CLP. Platelet transcriptional (ρ = 0.42, P = 3.2 × 10-285) and translational (ρ = 0.65, P = 1.09 × 10-56) changes were significantly correlated between septic patients and mice. We focused on ITGA2B, tracking and validating the expression, regulation, and functional impact of changes in ITGA2B during sepsis. Increased ITGA2B was identified in bone marrow megakaryocytes within 24 hours of sepsis onset. Subsequent increases in ITGA2B were seen in circulating platelets, suggesting dynamic trafficking of the messenger RNA. Transcriptional changes in ITGA2B were accompanied by de novo protein synthesis of αIIb and integrin αIIbß3 activation. Increased αIIb was associated with mortality in humans and mice. These findings provide previously unrecognized evidence that human and murine sepsis similarly alters the platelet transcriptional and translational landscape. Moreover, ITGA2B is upregulated and functional in sepsis due to trafficking from megakaryocytes and de novo synthesis in platelets and is associated with increased mortality.

Fibronectin modulates formation of PF4/heparin complexes and is a potential factor for reducing risk of developing HIT.

Heparin-induced thrombocytopenia (HIT) is caused by platelet-activating anti-platelet factor 4 (PF4)/heparin antibodies. Platelet activation assays that use "washed" platelets are more sensitive for detecting HIT antibodies than platelet-rich plasma (PRP)-based assays. Moreover, heparin-exposed patients vary considerably with respect to the risk of PF4/heparin immunization and, among antibody-positive patients, the risk of subsequent "breakthrough" of clinical HIT with manifestation of thrombocytopenia. We used washed platelets and PRP, standard laboratory HIT tests, and physicochemical methods to identify a plasma factor interfering with PF4/heparin complexes and anti-PF4/heparin antibody-platelet interaction, thus explaining differences in functional assays. To investigate a modulating risk for PF4/heparin immunization and breakthrough of HIT, we also tested 89 plasmas from 2 serosurveillance trials. Fibronectin levels were measured in 4 patient groups exhibiting different degrees of heparin-dependent immunization and expression of HIT. The heat-labile plasma protein, fibronectin, inhibited PF4 binding to platelets in a dose-dependent fashion, particularly in washed (vs PRP) systems. Fibronectin also inhibited PF4/heparin binding to platelets, anti-PF4/heparin antibody binding to PF4/heparin complexes, and anti-PF4/heparin antibody-induced platelet activation as a result of PF4/heparin complex disruption. In addition, plasma fibronectin levels increased progressively among the following 4 patient groups: enzyme-linked immunosorbent assay (ELISA)+/serotonin-release assay (SRA)+/HIT+ < ELISA+/SRA+/HIT- ∼ ELISA+/SRA-/HIT- < ELISA-/SRA-/HIT-. Altogether, these findings suggest that fibronectin interferes with PF4/heparin complex formation and anti-PF4/heparin antibody-induced platelet activation. Reduced fibronectin levels in washed platelet assays help to explain the greater sensitivity of washed platelet (vs PRP) assays for HIT. More importantly, lower plasma fibronectin levels could represent a risk factor for PF4/heparin immunization and clinical breakthrough of HIT.

Serotype 3 pneumococci sequester platelet-derived human thrombospondin-1 via the adhesin and immune evasion protein Hic.

Streptococcus pneumoniae serotype 3 strains emerge frequently within clinical isolates of invasive diseases. Bacterial invasion into deeper tissues is associated with colonization and immune evasion mechanisms. Thus, pneumococci express a versatile repertoire of surface proteins sequestering and interacting specifically with components of the human extracellular matrix and serum. Hic, a PspC-like pneumococcal surface protein, possesses vitronectin and factor H binding activity. Here, we show that heterologously expressed Hic domains interact, similar to the classical PspC molecule, with human matricellular thrombospondin-1 (hTSP-1). Binding studies with isolated human thrombospondin-1 and various Hic domains suggest that the interaction between hTSP-1 and Hic differs from binding to vitronectin and factor H. Binding of Hic to hTSP-1 is inhibited by heparin and chondroitin sulfate A, indicating binding to the N-terminal globular domain or type I repeats of hTSP-1. Competitive inhibition experiments with other pneumococcal hTSP-1 adhesins demonstrated that PspC and PspC-like Hic recognize similar domains, whereas PavB and Hic can bind simultaneously to hTSP-1. In conclusion, Hic binds specifically hTSP-1; however, truncation in the N-terminal part of Hic decreases the binding activity, suggesting that the full length of the α-helical regions of Hic is required for an optimal interaction.

Further insights into the anti-PF4/heparin IgM immune response.

Anti-platelet factor 4 (PF4)/heparin antibodies are not only the cause of heparin-induced thrombocytopenia but might also play a role in the antibacterial host defence. Recently, marginal zone (MZ) B cells were identified to be crucial for anti-PF4/heparin IgG antibody production in mice. Combining human studies and a murine model of polymicrobial sepsis we further characterised the far less investigated anti-PF4/heparin IgM immune response. We detected anti-PF4/heparin IgM antibodies in the sera of paediatric patients < 6 months of age after cardiac surgery and in sera of splenectomised mice subjected to polymicrobial sepsis. In addition, PF4/heparin-specific IgM B cells were not only found in murine spleen, but also in peritoneum and bone marrow upon in vitro stimulation. Together, this indicates involvement of additional B cell populations, as MZ B cells are not fully developed in humans until the second year of life and are restricted to the spleen in mice. Moreover, PF4/heparin-specific B cells were detected in human cord blood upon in vitro stimulation and PF4-/- mice produced anti-PF4/heparin IgM antibodies after polymicrobial sepsis. In conclusion, the anti-PF4/heparin IgM response is a potential innate immune reaction driven by a B cell population distinct from MZ B cells.

Partially desulfated heparin modulates the interaction between anti-protamine/heparin antibodies and platelets.

Protamine (PRT) is the standard drug to neutralise heparin. PRT/heparin complexes induce an immune response similar to that observed in heparin-induced thrombocytopenia (HIT). Partially desulfated heparin (ODSH) was shown to interfere with anti-platelet factor 4/heparin antibodies (Abs), which are responsible for HIT. In this study, we analyse the impact of ODSH on the interaction between anti-PRT/heparin Abs and platelets. The ability of ODSH to prevent anti-PRT/heparin Ab-induced platelet destruction in vivo was investigated using the NOD/SCID mouse model. ODSH improved platelet survival in the presence of PRT, heparin and anti-PRT/heparin Abs (median platelet survival after 300 minutes (min) with 20 µg/ml ODSH: 75%, range 70-81% vs without ODSH: 49%, range 44-59%, p=0.006). Furthermore, when ODSH was applied 60 min after Ab injection platelet survival was improved (median platelet survival after 300 min with ODSH: 83%, range 77-93% vs without ODSH: 59%, range 29-61%, p=0.02). In in vitro experiments ODSH inhibited platelet activation at concentrations >16 µg/mL (p<0.001), as well as PRT/heparin complex binding to platelets (mean fluorescence intensity [MFI] without ODSH: 85 ± 14 vs with ODSH: 15 ± 0.6, p=0.013). ODSH also displaced pre-bound complexes from the platelet surface (MFI without ODSH: 324 ± 43 vs with 32 µg/ml ODSH: 53 ± 9, p<0.001). While interfering with platelet activation by anti-PRT/heparin Abs, up to a concentration of 16 µg/ml, ODSH had only minimal impact on neutralisation of heparin by PRT. In conclusion, our study shows that ODSH is able to inhibit platelet activation and destruction suggesting a potential clinical use to reduce anti-PRT/heparin Ab-mediated adverse effects.

Polyphosphates form antigenic complexes with platelet factor 4 (PF4) and enhance PF4-binding to bacteria.

Short chain polyphosphates (polyP) are pro-coagulant and pro-inflammatory platelet released inorganic polymers. The platelet chemokine platelet factor 4 (PF4) binds to lipid A on bacteria, inducing an antibody mediated host defense mechanism, which can be misdirected against PF4/heparin complexes leading to the adverse drug reaction heparin-induced thrombocytopenia (HIT). Here, we demonstrate that PF4 complex formation with soluble short chain polyP contributes to host defense mechanisms. Circular dichroism spectroscopy and isothermal titration calorimetry revealed that PF4 changed its structure upon binding to polyP in a similar way as seen in PF4/heparin complexes. Consequently, PF4/polyP complexes exposed neoepitopes to which human anti-PF4/heparin antibodies bound. PolyP enhanced binding of PF4 to Escherichia coli, hereby facilitating bacterial opsonisation and, in the presence of human anti-PF4/polyanion antibodies, phagocytosis. Our study indicates a role of polyP in enhancing PF4-mediated defense mechanisms of innate immunity.

Pneumococcal Adhesins PavB and PspC Are Important for the Interplay with Human Thrombospondin-1.

The human matricellular glycoprotein thrombospondin-1 (hTSP-1) is released by activated platelets and mediates adhesion of Gram-positive bacteria to various host cells. In staphylococci, the adhesins extracellular adherence protein (Eap) and autolysin (Atl), both surface-exposed proteins containing repeating structures, were shown to be involved in the acquisition of hTSP-1 to the bacterial surface. The interaction partner(s) on the pneumococcal surface was hitherto unknown. Here, we demonstrate for the first time that pneumococcal adherence and virulence factor B (PavB) and pneumococcal surface protein C (PspC) are key players for the interaction of Streptococcus pneumoniae with matricellular hTSP-1. PavB and PspC are pneumococcal surface-exposed adhesins and virulence factors exhibiting repetitive sequences in their core structure. Heterologously expressed fragments of PavB and PspC containing repetitive structures exhibit hTSP-1 binding activity as shown by ELISA and surface plasmon resonance studies. Binding of hTSP-1 is charge-dependent and inhibited by heparin. Importantly, the deficiency in PavB and PspC reduces the recruitment of soluble hTSP-1 by pneumococci and decreases hTSP-1-mediated pneumococcal adherence to human epithelial cells. Platelet activation assays suggested that PavB and PspC are not involved in the activation of purified human platelets by pneumococci. In conclusion, this study indicates a pivotal role of PavB and PspC for pneumococcal recruitment of soluble hTSP-1 to the bacterial surface and binding of pneumococci to host cell-bound hTSP-1 during adhesion.

Binding of anti-platelet factor 4/heparin antibodies depends on the thermodynamics of conformational changes in platelet factor 4.

The chemokine platelet factor 4 (PF4) undergoes conformational changes when complexing with polyanions. This can induce the antibody-mediated adverse drug effect of heparin-induced thrombocytopenia (HIT). Understanding why the endogenous protein PF4 becomes immunogenic when complexing with heparin is important for the development of other negatively charged drugs and may also hint toward more general mechanisms underlying the induction of autoantibodies to other proteins. By circular dichroism spectroscopy, atomic force microscopy, and isothermal titration calorimetry we characterized the interaction of PF4 with unfractionated heparin (UFH), its 16-, 8-, and 6-mer subfractions, low-molecular-weight heparin (LMWH), and the pentasaccharide fondaparinux. To bind anti-PF4/heparin antibodies, PF4/heparin complexes require (1) an increase in PF4 antiparallel ß-sheets exceeding ∼30% (achieved by UFH, LMWH, 16-, 8-, 6-mer), (2) formation of multimolecular complexes (UFH, 16-, 8-mer), and (3) energy (needed for a conformational change), which is released by binding of ≥11-mer heparins to PF4, but not by smaller heparins. These findings may help to synthesize safer heparins. Beyond PF4 and HIT, the methods applied in the current study may be relevant to unravel mechanisms making other endogenous proteins more vulnerable to undergo conformational changes with little energy requirement (eg, point mutations and post-translational modifications) and thereby predisposing them to become immunogenic.