Cellular Components Contributing to the Development of Venous Thrombosis in Patients with Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive type of cancer and has a poor prognosis. Patients with PDAC are at high risk of developing thromboembolic events, which is a leading cause of morbidity and mortality following cancer progression. Plasma-derived coagulation is the most studied process in cancer-associated thrombosis. Other blood components, such as platelets, red blood cells, and white blood cells, have been gaining less attention. This narrative review addresses the literature on the role of cellular components in the development of venous thromboembolism (VTE) in patients with PDAC. Blood cells seem to play an important role in the development of VTE. Altered blood cell counts, i.e., leukocytosis, thrombocytosis, and anemia, have been found to associate with VTE risk. Tumor-related activation of leukocytes leads to the release of tissue factor-expressing microvesicles and the formation of neutrophil extracellular traps, initiating coagulation and forming a scaffold for thrombi. Tissue factor-expressing microvesicles are also thought to be released by PDAC cells. PDAC cells have been shown to stimulate platelet activation and aggregation, proposedly via the secretion of podoplanin and mucins. Hypofibrinolysis, partially explained by increased plasminogen activator inhibitor-1 activity, is observed in PDAC. In short, PDAC-associated hypercoagulability is a complex and multifactorial process. A better understanding of cellular contributions to hypercoagulability might lead to the improvement of diagnostic tests to identify PDAC patients at highest risk of VTE.

Nonredundant Roles of Platelet Glycoprotein VI and Integrin αIIbß3 in Fibrin-Mediated Microthrombus Formation.

OBJECTIVE: Fibrin is considered to strengthen thrombus formation via integrin αIIbß3, but recent findings indicate that fibrin can also act as ligand for platelet glycoprotein VI. Approach and Results: To investigate the thrombus-forming potential of fibrin and the roles of platelet receptors herein, we generated a range of immobilized fibrin surfaces, some of which were cross-linked with factor XIIIa and contained VWF-BP (von Willebrand factor-binding peptide). Multicolor microfluidics assays with whole-blood flowed at high shear rate (1000 s-1) indicated that the fibrin surfaces, regardless of the presence of factor XIIIa or VWF-BP, supported platelet adhesion and activation (P-selectin expression), but only microthrombi were formed consisting of bilayers of platelets. Fibrinogen surfaces produced similar microthrombi. Markedly, tiggering of coagulation with tissue factor or blocking of thrombin no more than moderately affected the fibrin-induced microthrombus formation. Absence of αIIbß3 in Glanzmann thrombasthenia annulled platelet adhesion. Blocking of glycoprotein VI with Fab 9O12 substantially, but incompletely reduced platelet secretion, Ca2+ signaling and aggregation, while inhibition of Syk further reduced these responses. In platelet suspension, glycoprotein VI blockage or Syk inhibition prevented fibrin-induced platelet aggregation. Microthrombi on fibrin surfaces triggered only minimal thrombin generation, in spite of thrombin binding to the fibrin fibers. CONCLUSIONS: Together, these results indicate that fibrin fibers, regardless of their way of formation, act as a consolidating surface in microthrombus formation via nonredundant roles of platelet glycoprotein VI and integrin αIIbß3 through signaling via Syk and low-level Ca2+ rises.

Flow Cytometry Based Platelet Reactivity Testing to Predict the Occurrence of Per-operative Solid Microemboli During Carotid Endarterectomy.

OBJECTIVE: Peri-operative antiplatelet therapy (APT) aims to prevent thrombotic events such as stroke. High platelet reactivity ,despite the use use of APT, increases the risk of thrombotic events. Transcranial Doppler imaging (TCD) is used to detect peri-operative microembolic signals (MES) during carotid endarterectomy (CEA). Peri-operative MES are associated with an increased risk of procedural stroke and new silent lesions on diffusion weighted magnetic resonance imaging following surgery. The main components of TCD detected MES are platelet aggregates, and therefore patients displaying multiple MES during surgery could have benefited from more stringent APT. This study investigated whether the use of flow cytometry based platelet reactivity measurements were correlated with the incidence of pre-operative MES and thereby in the future suitable to predict patients at increased risk of peri-operative thrombotic events. METHODS: Bilateral TCD with MES detection was performed in 197 patients undergoing CEA. Platelet reactivity was assessed with a flow cytometry based platelet reactivity assay measuring platelet response in whole blood. High on treatment platelet reactivity status was assessed for all patients. The secondary outcome was major adverse cardiovascular events (MACE) within one year. RESULTS: In total, 197 patients were included, 49 had peri-operative MES. The platelet response to adenosine diphosphate (ADP) correlated with MES (p = .021), and high on treatment platelet reactivity after adenosine diphosphate stimulation was associated with MACE (OR 2.34, 95% confidence interval 1.126 - 4.890, p = .023). CONCLUSION: Pre-operative platelet reactivity determined by flow cytometry after ADP stimulation correlated with the occurrence of intra-operative MES and post-operative MACE. Clopidogrel treatment showed the most substantial effect on reducing MES frequency and platelet reactivity measured by flow cytometry.

Reversible Platelet Integrin αIIbß3 Activation and Thrombus Instability.

Integrin αIIbß3 activation is essential for platelet aggregation and, accordingly, for hemostasis and arterial thrombosis. The αIIbß3 integrin is highly expressed on platelets and requires an activation step for binding to fibrinogen, fibrin or von Willebrand factor (VWF). A current model assumes that the process of integrin activation relies on actomyosin force-dependent molecular changes from a bent-closed and extended-closed to an extended-open conformation. In this paper we review the pathways that point to a functional reversibility of platelet αIIbß3 activation and transient aggregation. Furthermore, we refer to mouse models indicating that genetic defects that lead to reversible platelet aggregation can also cause instable thrombus formation. We discuss the platelet agonists and signaling pathways that lead to a transient binding of ligands to integrin αIIbß3. Our analysis points to the (autocrine) ADP P2Y1 and P2Y12 receptor signaling via phosphoinositide 3-kinases and Akt as principal pathways linked to reversible integrin activation. Downstream signaling events by protein kinase C, CalDAG-GEFI and Rap1b have not been linked to transient integrin activation. Insight into the functional reversibility of integrin activation pathways will help to better understand the effects of antiplatelet agents.

Von Willebrand factor, ADAMTS13 and mortality in dialysis patients.

BACKGROUND: Von Willebrand Factor (VWF) multimers are cleaved into smaller and less coagulant forms by the metalloprotease ADAMTS13. The aim of this study was to investigate the association between VWF and ADAMTS13 and mortality in dialysis patients. METHODS: We prospectively followed 956 dialysis patients. VWF levels and ADAMTS13 activity were measured. Cox proportional hazard analyses were used to calculate hazard ratios (HRs) with 95 % confidence intervals (CIs) to investigate the association between quartiles of VWF levels and ADAMTS13 activity and all-cause mortality. HRs were adjusted for age, sex, body mass index, cardiovascular disease, dialysis modality, primary kidney disease, use of antithrombotic medication, systolic blood pressure, albumin, C-reactive protein and residual GFR. RESULTS: Of the 956 dialysis patients, 288 dialysis patients died within three years (mortality rate 151 per 1000 person-years). The highest quartile of VWF as compared with lower levels of VWF was associated with a 1.4-fold (95 %CI 1.1-1.8) increased mortality risk after adjustment. The lowest quartile of ADAMTS13 activity as compared with other quartiles was associated with a 1.3-fold (95 %CI 1.0-1.7) increased mortality risk after adjustment. The combination of the highest VWF quartile and lowest ADAMTS13 quartile was associated with a 2.0-fold (95 %CI 1.3-3.0) increased mortality risk as compared with the combination of the lowest VWF quartile and highest ADAMTS13 quartile. CONCLUSIONS: High VWF levels and low ADAMTS13 activity were associated with increased mortality risks in dialysis patients.

Platelet function is disturbed by the angiogenesis inhibitors sunitinib and sorafenib, but unaffected by bevacizumab.

INTRODUCTION: At the clinical introduction of antiangiogenic agents as anticancer agents, no major toxicities were expected as merely just endothelial cells (ECs) in tumors would be affected. However, several (serious) toxicities became apparent, of which underlying mechanisms are largely unknown. We investigated to what extent sunitinib (multitargeted antiangiogenic tyrosine kinase inhibitor (TKI)), sorafenib (TKI) and bevacizumab [specific antibody against vascular endothelial growth factor (VEGF)] may impair platelet function, which might explain treatment-related bleedings. MATERIALS AND METHODS: In vitro, the influence of sunitinib, sorafenib, and bevacizumab on platelet aggregation, P-selectin expression and fibrinogen binding, platelet-EC interaction, and tyrosine phosphorylation of c-Src was studied by optical aggregation, flow cytometry, real-time perfusion, and western blotting. Ex vivo, platelet aggregation was analyzed in 25 patients upon sunitinib or bevacizumab treatment. Concentrations of sunitinib, VEGF, and platelet and EC activation markers were measured by LC-MS/MS and ELISA. RESULTS: In vitro, sunitinib and sorafenib significantly inhibited platelet aggregation (20 µM sunitinib: 71.3%, p < 0.001; 25 µM sorafenib: 55.8%, p = 0.042). Sorafenib and sunitinib significantly inhibited P-selectin expression on platelets. Exposure to both TKIs resulted in a reduced tyrosine phosphorylation of c-Src. Ex vivo, within 24 h sunitinib impaired platelet aggregation (83.0%, p = 0.001, N = 8). Plasma concentrations of sunitinib, VEGF, and platelet/EC activation markers were not correlated with disturbed aggregation. In contrast, bevacizumab only significantly impaired platelet aggregation in vitro at high concentrations, but not ex vivo. CONCLUSION: Sunitinib significantly inhibits platelet aggregation in patients already after 24 h of first administration, whereas bevacizumab had no effect on aggregation. These findings may explain the clinically observed bleedings during treatment with antiangiogenic TKIs.

Effects of Repeated Bouts of Exercise on the Hemostatic System.

Physical activity is beneficial for health, for example, by lowering the risk of cardiovascular events. However, vigorous exercise is associated with the occurrence of thromboembolic events and sudden cardiac death, in particular in untrained individuals. Whereas acute exercise is known to cause a hypercoagulable state, repeated exposure to (strenuous) exercise by means of training may actually condition the hemostatic response to exercise. To date, the effects of exercise training on blood coagulability and the underlying mechanisms have yet to be fully discerned. In this review, the authors provide an overview of existing literature on how training programs and training status influence hemostasis in healthy individuals. Furthermore, they present data of a pilot study in which we studied the effects of repetitive submaximal intensity cycling on procoagulant and anticoagulant processes. It is known that factor VIII (FVIII) and von Willebrand factor (VWF) increase after exercise, but we found that this increase in FVIII and VWF (antigen, propeptide, and VWF in active conformation) was smaller on each of three subsequent days, suggesting either adaptation of endothelial activation or exhaustion of endothelial VWF supplies. With respect to thrombin generation, elevated FVIII significantly increased the thrombin generation peak but not the endogenous thrombin potential. In contrast, platelet activation in terms of P-selectin expression after stimulation with protease-activated receptor-1 and glycoprotein VI agonists decreased after exercise and did not recover, indicating exhaustion of the platelet response to repetitive exercise.

Proteome-wide analysis and CXCL4 as a biomarker in systemic sclerosis.

BACKGROUND: Plasmacytoid dendritic cells have been implicated in the pathogenesis of systemic sclerosis through mechanisms beyond the previously suggested production of type I interferon. METHODS: We isolated plasmacytoid dendritic cells from healthy persons and from patients with systemic sclerosis who had distinct clinical phenotypes. We then performed proteome-wide analysis and validated these observations in five large cohorts of patients with systemic sclerosis. Next, we compared the results with those in patients with systemic lupus erythematosus, ankylosing spondylitis, and hepatic fibrosis. We correlated plasma levels of CXCL4 protein with features of systemic sclerosis and studied the direct effects of CXCL4 in vitro and in vivo. RESULTS: Proteome-wide analysis and validation showed that CXCL4 is the predominant protein secreted by plasmacytoid dendritic cells in systemic sclerosis, both in circulation and in skin. The mean (±SD) level of CXCL4 in patients with systemic sclerosis was 25,624±2652 pg per milliliter, which was significantly higher than the level in controls (92.5±77.9 pg per milliliter) and than the level in patients with systemic lupus erythematosus (1346±1011 pg per milliliter), ankylosing spondylitis (1368±1162 pg per milliliter), or liver fibrosis (1668±1263 pg per milliliter). CXCL4 levels correlated with skin and lung fibrosis and with pulmonary arterial hypertension. Among chemokines, only CXCL4 predicted the risk and progression of systemic sclerosis. In vitro, CXCL4 down-regulated expression of transcription factor FLI1, induced markers of endothelial-cell activation, and potentiated responses of toll-like receptors. In vivo, CXCL4 induced the influx of inflammatory cells and skin transcriptome changes, as in systemic sclerosis. CONCLUSIONS: Levels of CXCL4 were elevated in patients with systemic sclerosis and correlated with the presence and progression of complications, such as lung fibrosis and pulmonary arterial hypertension. (Funded by the Dutch Arthritis Association and others.).

The effect of P2Y12 inhibition on platelet activation assessed with aggregation- and flow cytometry-based assays.

Patients on P2Y12 inhibitors may still develop thrombosis or bleeding complications. Tailored antiplatelet therapy, based on platelet reactivity testing, might reduce these complications. Several tests have been used, but failed to show a benefit of tailored antiplatelet therapy. This could be due to the narrowness of current platelet reactivity tests, which are limited to analysis of platelet aggregation after stimulation of the adenosine diphosphate (ADP)-pathway. However, the response to ADP does not necessarily reflect the effect of P2Y12 inhibition on platelet function in vivo. Therefore, we investigated whether measuring platelet reactivity toward other physiologically relevant agonists could provide more insight in the efficacy of P2Y12 inhibitors. The effect of in vitro and in vivo P2Y12 inhibition on αIIbß3-activation, P-selectin and CD63-expression, aggregate formation, release of alpha, and dense granules content was assessed after stimulation of different platelet activation pathways. Platelet reactivity measured with flow cytometry in 72 patients on P2Y12 inhibitors was compared to VerifyNow results. P2Y12 inhibitors caused strongly attenuated platelet fibrinogen binding after stimulation with peptide agonists for protease activated receptor (PAR)-1 and -4, or glycoprotein VI ligand crosslinked collagen-related peptide (CRP-xl), while aggregation was normal at high agonist concentration. P2Y12 inhibitors decreased PAR-agonist and CRP-induced dense granule secretion, but not alpha granule secretion. A proportion of P2Y12-inhibitor responsive patients according to VerifyNow, displayed normal fibrinogen binding assessed with flow cytometry after stimulation with PAR-agonists or CRP despite full inhibition of the response to ADP, indicating suboptimal platelet inhibition. Concluding, measurement of platelet fibrinogen binding with flow cytometry after stimulation of thrombin- or collagen receptors in addition to ADP response identifies different patients as nonresponders to P2Y12 inhibitors, compared to only ADP-induced aggregation-based assays. Future studies should investigate the value of both assays for monitoring on-treatment platelet reactivity.

Risk factor analysis of cerebral white matter hyperintensities in children with sickle cell disease.

Sickle cell disease (SCD) is complicated by silent cerebral infarcts, visible as white matter hyperintensities (WMHs) on magnetic resonance imaging (MRI). Both local vaso-occlusion, elicited by endothelial dysfunction, and insufficiency of cerebral blood flow (CBF) have been proposed to be involved in the aetiology. We performed an explorative study to investigate the associations between WMHs and markers of endothelial dysfunction and CBF by quantifying WMH volume on 3.0 Tesla MRI. We included 40 children with HbSS or HbSß(0) thalassaemia, with a mean age of 12.1 ± 2.6 years. Boys demonstrated an increased risk for WMHs (odds ratio 4.5, 95% confidence interval 1.2-17.4), unrelated to glucose-6-phosphate dehydrogenase deficiency. In patients with WMHs, lower fetal haemoglobin (HbF) was associated with a larger WMH volume (regression coefficient = -0.62, R2 = 0.5, P = 0.04). Lower ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) levels were associated with lower CBF in the white matter (regression coefficient = 0.07, R2 = 0.15, P = 0.03), suggesting that endothelial dysfunction could potentially hamper CBF. The findings of our explorative study suggest that a high level of HbF may be protective for WMHs and that endothelial dysfunction may contribute to the development of WMHs by reducing CBF.

Active von Willebrand factor predicts 28-day mortality in patients with systemic inflammatory response syndrome.

Endothelial dysfunction contributes to the pathology of systemic inflammatory response syndrome (SIRS). However, endothelial biomarkers are not routinely evaluated in this setting. Here, 275 patients with SIRS and plasma levels of von Willebrand factor (VWF), thrombospondin-1, myeloperoxidase, ADAMTS-13, and active VWF (aVWF) were studied in relation to 28-day mortality. On admission, aVWF levels were higher in nonsurvivors vs survivors (0.69 vs 0.47 µg/mL, P = .019). Patients in the highest tertile of aVWF levels had a lower cumulative survival (86% vs 75%, P = .017) and twofold increased hazard ratio (HR). When adjusted for the Acute Physiology and Chronic Health Evaluation IV (APACHE-IV) score, this difference remained significant (HR 1.82, 95% confidence interval, 1.03-3.3). On admission, no significant differences were measured for the other proteins. These observations suggest that the stimulated release of VWF is not predictive for mortality in patients with SIRS, opposite of the processing of VWF after release. aVWF could be used with the APACHE-IV score to stratify SIRS patients at high mortality risk.

Functional platelet defects in children with severe chronic ITP as tested with 2 novel assays applicable for low platelet counts.

Immune thrombocytopenia (ITP) is an autoimmune disease with a complex heterogeneous pathogenesis and a bleeding phenotype that is not necessarily correlated to platelet count. In this study, the platelet function was assessed in a well-defined cohort of 33 pediatric chronic ITP patients. Because regular platelet function test cannot be performed in patients with low platelet counts, 2 new assays were developed to determine platelet function: first, the microaggregation test, measuring in platelets isolated from 10 mL of whole blood the platelet potential to form microaggregates in response to an agonist; second, the platelet reactivity assay, measuring platelet reactivity to adenosine diphosphate (ADP), convulxin (CVX), and thrombin receptor activator peptide in only 150 µL of unprocessed whole blood. Patients with a severe bleeding phenotype demonstrated a decreased aggregation potential upon phorbol myristate acetate stimulation, decreased platelet degranulation following ADP stimulation, and a higher concentration of ADP and CVX needed to activate the glycoprotein IIbIIIa complex compared with patients with a mild bleeding phenotype. In conclusion, here we have established 2 functional tests that allow for evaluation of platelet function in patients with extremely low platelet counts (<10(9)). These tests show that platelet function is related to bleeding phenotype in chronic ITP.

Functional recovery of stored platelets after transfusion.

BACKGROUND: Platelet (PLT) concentrates are prophylactically given to prevent major bleeding complications. The corrected count increment (CCI) is currently the only tool to monitor PLT transfusion efficacy. PLT function tests cannot be performed in patients with thrombocytopenia. Therefore, an optimized agonist-induced assay was used to determine PLT function, in patients with severe thrombocytopenia before and after transfusion. STUDY DESIGN AND METHODS: PLT reactivity toward adenosine diphosphate (ADP), thrombin receptor-activating peptide SFLLRN (TRAP), and convulxin (CVX) was assessed by flow cytometry. P-selectin expression was measured on PLTs from 11 patients with thrombocytopenia before and 1 hour after transfusion, on stored PLTs, and on stored PLTs incubated for 1 hour in whole blood from patients ex vivo. RESULTS: The mean (±SEM) CCI after 1 hour was 11.4 (±1.5). After transfusion, maximal agonist-induced PLT P-selectin expression was on average 29% higher for ADP (p = 0.02), 25% higher for TRAP (p = 0.007), and 24% higher for CVX (p = 0.0008). ADP-induced reactivity of stored PLTs increased with 46% after ex vivo incubation (p = 0.007). These PLTs also showed an overall higher P-selectin expression compared to PLTs 1 hour after transfusion (p = 0.005). After normalization for this background expression, a similar responsiveness was observed. CONCLUSIONS: Our study shows recovery of PLT function after transfusion in patients with thrombocytopenia. The majority of functional PLTs measured after transfusion most likely represents stored transfused PLTs that regained functionality in vivo. The difference in baseline P-selectin expression in vivo versus ex vivo suggests a rapid clearance from circulation of PLTs with increased P-selectin expression.

FLow-induced PRotrusions (FLIPRs): a platelet-derived platform for the retrieval of microparticles by monocytes and neutrophils.

RATIONALE: Platelets are the most important cells in the primary prevention of blood loss after injury. In addition, platelets are at the interface between circulating leukocytes and the (sub)endothelium regulating inflammatory responses. OBJECTIVE: Our aim was to study the dynamic process that leads to the formation of procoagulant and proinflammatory platelets under physiological flow. METHODS AND RESULTS: In the present study, we describe the formation of extremely long, negatively charged membrane strands that emerge from platelets adhered under flow. These flow-induced protrusions (FLIPRs) are formed in vitro on different physiological substrates and are also detected in vivo in a mouse carotid injury model. FLIPRs are formed downstream the adherent and activated platelets and reach lengths of 250 µm. FLIPR formation is shear-dependent and requires cyclophilin D, calpain, and Rac1 activation. It is accompanied by a disassembly of the F-actin and microtubule organization. Monocytes and neutrophils roll over FLIPRs in a P-selectin/P-selectin glycoprotein ligand-1-dependent manner, retrieving fragments of FLIPRs as microparticles on their surface. Consequently, monocytes and neutrophils become activated, as demonstrated by increased CD11b expression and L-selectin shedding. CONCLUSIONS: The formation of long platelet membrane extensions, such as the ones presented in our flow model, may pave the way to generate an increased membrane surface for interaction with monocytes and neutrophils. Our study provides a mechanistic model for platelet membrane transfer and the generation of monocyte/neutrophil-microparticle complexes. We propose that the formation of FLIPRs in vivo contributes to the well-established proinflammatory function of platelets and platelet-derived microparticles.

Plasmin cleavage of von Willebrand factor as an emergency bypass for ADAMTS13 deficiency in thrombotic microangiopathy.

BACKGROUND: Von Willebrand factor (VWF) multimer size is controlled through continuous proteolysis by ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type I motif, member 13). This prevents spontaneous platelet agglutination and microvascular obstructions. ADAMTS13 deficiency is associated with thrombotic thrombocytopenic purpura, in which life-threatening episodes of microangiopathy damage kidneys, heart, and brain. Enigmatically, a complete ADAMTS13 deficiency does not lead to continuous microangiopathy. We hypothesized that plasmin, the key enzyme of the fibrinolytic system, serves as a physiological backup enzyme for ADAMTS13 in the degradation of pathological platelet-VWF complexes. METHODS AND RESULTS: Using real-time microscopy, we determined that plasmin rapidly degrades platelet-VWF complexes on endothelial cells in absence of ADAMTS13, after activation by urokinase-type plasminogen activator or the thrombolytic agent streptokinase. Similarly, plasmin degrades platelet-VWF complexes in platelet agglutination studies. Plasminogen directly binds to VWF and its A1 domain in a lysine-dependent manner, as determined by enzyme-linked immunosorbent assay. Plasma levels of plasmin-α2-antiplasmin complexes increase with the extent of thrombocytopenia in patients with acute episodes of thrombotic thrombocytopenic purpura, independent of ADAMTS13 activity. This indicates that plasminogen activation takes place during microangiopathy. Finally, we show that the thrombolytic agent streptokinase has therapeutic value for Adamts13(-/-) mice in a model of thrombotic thrombocytopenic purpura. CONCLUSIONS: We propose that plasminogen activation on endothelial cells acts as a natural backup for ADAMTS13 to degrade obstructive platelet-VWF complexes. Our findings indicate that thrombolytic agents may have therapeutic value in the treatment of microangiopathies and may be useful to bypass inhibitory antibodies against ADAMTS13 that cause thrombotic thrombocytopenic purpura.

Hemocompatibility assessment of two siRNA nanocarrier formulations.

PURPOSE: Since the discovery of RNAi and its therapeutic potential, carrier systems have been developed to deliver small RNAs (particularly siRNA) for modulation of gene expression at the post-transcriptional level. An important factor determining the fate and usability of these systems in vivo is interaction with blood components, blood cells, and the immune system. In this study, a lipid-based and a polymer-based carrier system containing siRNA have been investigated in vitro in terms of their hemocompatibility. METHODS: The nanocomplexes studied were Angiplex, a targeted lipid-based system, and pHPMA-MPPM polyplex, a formulation based on a cationic polymer. siVEGFR-2 was encapsulated in both carriers and activation of platelets, coagulation, and complement cascade as well as induction of platelet aggregation were evaluated in vitro. RESULTS: Both systems had been shown before to cause significant silencing in vitro. Our findings indicated that pHPMA-MPPM polyplex triggered high platelet activation and aggregation although it did not stimulate coagulation substantially. Angiplex, on the other hand, provoked insignificant activation and aggregation of platelets and activated coagulation minimally. Complement system activation by Angiplex was in general low but stronger than pHPMA-MPPM polyplex. CONCLUSIONS: Taken together, these in vitro assays may help the selection of suitable carriers for systemic delivery of siRNA in early preclinical investigations and reduce the use of laboratory animals significantly.

High precision platelet releasate definition by quantitative reversed protein profiling--brief report.

OBJECTIVE: Platelet activation and subsequent protein release play an important role in healthy hemostasis and inflammatory responses, yet the identity and quantity of proteins in the platelet releasate are still debated. Here, we present a reversed releasate proteomics approach to determine unambiguously and quantitatively proteins released from activated platelets. APPROACH AND RESULTS: Isolated platelets were mock and fully stimulated after which the released proteins in the supernatant were removed. Using high-end proteomics technology (2D chromatography, stable isotope labeling, electron transfer dissociation, and high collision dissociation fragmentation) allowed us to quantitatively discriminate the released proteins from uncontrolled lysis products. Monitoring the copy numbers of ≈ 4500 platelet proteins, we observed that after stimulation via thrombin and collagen, only 124 (<3%) proteins were significantly released (P<0.05). The released proteins span a concentration range of ≥ 5 orders, as confirmed by ELISA. The released proteins were highly enriched in secretion tags and contained all known factors at high concentrations (>100 ng/mL, eg, thrombospondin, von Willebrand factor, and platelet factor 4). Interestingly, in the lower concentration range of the releasate many novel factors were identified. CONCLUSIONS: Our reversed releasate dataset forms the first unambiguous, in depth repository for molecular factors released by platelets.

Targeted phosphotyrosine profiling of glycoprotein VI signaling implicates oligophrenin-1 in platelet filopodia formation.

OBJECTIVE: Platelet adhesion to subendothelial collagen is dependent on the integrin α2ß1 and glycoprotein VI (GPVI) receptors. The major signaling routes in collagen-dependent platelet activation are outlined; however, crucial detailed knowledge of the actual phosphorylation events mediating them is still limited. Here, we explore phosphotyrosine signaling events downstream of GPVI with site-specific detail. APPROACH AND RESULTS: Immunoprecipitations of phosphotyrosine-modified peptides from protein digests of GPVI-activated and resting human platelets were compared by stable isotope-based quantitative mass spectrometry. We surveyed 214 unique phosphotyrosine sites over 2 time points, of which 28 showed a significant increase in phosphorylation on GPVI activation. Among these was Tyr370 of oligophrenin-1 (OPHN1), a Rho GTPase-activating protein. To elucidate the function of OPHN1 in platelets, we performed an array of functional platelet analyses within a small cohort of patients with rare oligophrenia. Because of germline mutations in the OPHN1 gene locus, these patients lack OPHN1 expression entirely and are in essence a human knockout model. Our studies revealed that among other unaltered properties, patients with oligophrenia show normal P-selectin exposure and αIIbß3 activation in response to GPVI, as well as normal aggregate formation on collagen under shear conditions. Finally, the major difference in OPHN1-deficient platelets turned out to be a significantly reduced collagen-induced filopodia formation. CONCLUSIONS: In-depth phosphotyrosine screening revealed many novel signaling recipients downstream of GPVI activation uncovering a new level of detail within this important pathway. To illustrate the strength of such data, functional follow-up of OPHN1 in human platelets deficient in this protein showed reduced filopodia formation on collagen, an important parameter of platelet hemostatic function.

The influence of Erythropoietin on platelet activation, thrombin generation and FVII/active FVII in patients with AMI.

BACKGROUND: Erythropoietin (Epo) has been shown to improve myocardial function in models of experimental myocardial infarction, but has also been associated with a rise in thromboembolic events. Thus, the aim of this study was to investigate the influence of Epo on platelet activation and coagulation in patients with acute myocardial infarction (AMI). METHODS: The study was designed as a substudy of the randomised, double-blind, placebo controlled REVIVAL-3 (REgeneration of VItal Myocardium in ST-Segment EleVation MyocardiAL Infarction by Erythropoietin) study that investigated the effects of recombinant human Epo in AMI. Serial venous blood samples were collected before and after study medication. Circulating prothrombin fragment F1 + 2, FVII, active FVII, beta thromboglobulin (TG) and P-Selectin were measured before and 60 hours after randomization by immunoassay (n = 94). In a randomly selected subgroup platelet aggregation was measured using whole blood aggregometry (Multiplate Analyzer, n = 45). RESULTS: After 5 days an increase in FVII was observed after Epo as compared to placebo (P = 0.02), yet active FVII and prothrombin fragment F1 + 2 remained unchanged. Moreover, no statistically significant differences in circulating TG or P-selectin were observed between the groups. As an expected response to peri-interventional therapy with clopidogrel and aspirin, platelet aggregation after stimulation with ADP, TRAP, ASPI or collagen decreased 12 hours and 2 days after PCI. However, no difference between the Epo and the placebo group was observed. CONCLUSION: After treatment with Epo in patients with AMI a slight increase in circulating FVII after Epo was not associated with an increase in active FVII, prothrombin fragment F1 + 2, TG or P-selectin. Moreover, platelet aggregation was not altered after treatment with Epo as compared to placebo. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01761435.