Arjan D. Barendrecht

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–&agr;2-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.

Quantitative proteomics analysis reveals similar release profiles following specific PAR-1 or PAR-4 stimulation of platelets

AIMSnPlatelets are a natural source of growth factors, cytokines and chemokines, that regulate angiogenesis and inflammation. It has been suggested that differential release of pro- and anti-angiogenic growth factors from platelet α-granules by protease-activated receptors (PAR) 1 and 4 may be important for the regulation of angiogenesis. We aimed to compare the releasates of unstimulated platelets with PAR-1- and PAR-4-stimulated platelets.nnnMETHODS AND RESULTSnThe release of β-thromboglobulin, platelet factor (PF)-4, thrombospondin, platelet-derived growth factor (PDGF)-A/B, regulated and normal T-cell expressed and secreted (RANTES/CCL5), endostatin, CXCL12, and vascular endothelial growth factor (VEGF) was measured with enzyme-linked immunosorbent assay (ELISA). Mass spectrometry (MS)-based quantitative proteomics identified 93 proteins from platelets stimulated with PAR-1 and PAR-4. A strong correlation between the factors released after either stimulus was observed (Spearmans r 0.94, P < 0.001). Analysis with ELISA showed that stimulation with PAR-1 or PAR-4 lead to non-differential release of β-thromboglobulin, PF-4, thrombospondin, PDGF-A/B, RANTES/CCL5, endostatin, CXCL12, and VEGF. Release of thrombospondin was slightly lower after PAR-1 stimulation (7.2 μg/mL), compared with PAR-4 induced release (9.8 μg/mL; P < 0.05).nnnCONCLUSIONSnBoth ELISA on established α-granule proteins and MS-based quantitative proteomics showed that the most abundant α-granule proteins are released in similar quantities from platelets after stimulation with either PAR-1 or PAR-4. Our findings provide evidence against the hypothesis that PAR-1 and PAR-4 stimulation of platelets trigger differential release of alpha-granule, but further studies are needed to draw conclusions for physiological conditions.

High platelet reactivity is associated with myocardial infarction in premenopausal women: a population-based case–control study

Summary.u2002 Background: Platelets are involved in the occlusion of coronary arteries after rupture of an atherosclerotic plaque. Furthermore, activated platelets release large quantities of growth factors, chemokines and interleukines that regulate inflammatory reactions. Therefore, we hypothesized that high basal platelet reactivity may contribute to an increased risk of myocardial infarction (MI) in premenopausal women. Methods: We assessed the relation between high platelet reactivity and MI in a population‐based case–control study among premenopausal women (aged <u200350u2003years). We used multivariable logistic regression to quantify the effect of high platelet reactivity, adjusted for potential confounders. Platelet reactivity was estimated by plasma levels of neutrophil activating peptide 2 (NAP‐2), CXC chemokine ligand (CXCL)4, soluble glycoprotein 1b (sGPIb) and soluble P‐selectin. Results: High platelet reactivity (i.e. levels ≥u200390th percentile control subjects) was associated with a 2‐ to 3‐fold increased incidence of MI: the adjusted odds ratios (ORs) were 3.0 [95% confidence interval (CI) 1.4–6.4] for NAP‐2, 2.2 (0.9–5.1) for CXCL4, 1.9 (0.7–4.6) for sP‐selectin and 2.5 (1.1–5.7) for sGPIb. The incidence of MI dose‐dependently increased when more markers were elevated. High platelet reactivity according to both NAP‐2 and sGPIb was associated with an up to tenfold increased incidence (9.9, 95% confidence interval 2.0–48.3). Conclusions: High basal platelet reactivity was associated with a 2‐ to 3‐fold higher incidence of MI compared with normal platelet reactivity in premenopausal women. Our results suggest that high basal platelet reactivity may contribute to a higher risk of MI.

Polyphosphate nanoparticles on the platelet surface trigger contact system activation

Polyphosphate is an inorganic polymer that can potentiate several interactions in the blood coagulation system. Blood platelets contain polyphosphate, and the secretion of platelet-derived polyphosphate has been associated with increased thrombus formation and activation of coagulation factor XII. However, the small polymer size of secreted platelet polyphosphate limits its capacity to activate factor XII in vitro. Thus, the mechanism by which platelet polyphosphate contributes to thrombus formation remains unclear. Using live-cell imaging, confocal and electron microscopy, we show that activated platelets retain polyphosphate on their cell surface. The apparent polymer size of membrane-associated polyphosphate largely exceeds that of secreted polyphosphate. Ultracentrifugation fractionation experiments revealed that membrane-associated platelet polyphosphate is condensed into insoluble spherical nanoparticles with divalent metal ions. In contrast to soluble polyphosphate, membrane-associated polyphosphate nanoparticles potently activate factor XII. Our findings identify membrane-associated polyphosphate in a nanoparticle state on the surface of activated platelets. We propose that these polyphosphate nanoparticles mechanistically link the procoagulant activity of platelets with the activation of coagulation factor XII.

Plasma concentration of von Willebrand factor predicts mortality in patients on chronic renal replacement therapy

BACKGROUNDnTraditional cardiovascular risk factors do not explain the high incidence of cardiovascular mortality and morbidity in patients with end-stage renal disease. A prothrombotic state could accelerate the process of vascular disease in these patients.nnnMETHODSnIn this study, four platelet activation markers (NAP-2, P-selectin, GP1b and RANTES) and two endothelial cell activation markers (von Willebrand factor and its propeptide) were measured in 671 haemodialysis patients and 275 patients on continuous ambulatory peritoneal dialysis (PD). All were long-term dialysis patients. The risk of all-cause and cardiovascular mortality was assessed in relation to these markers after a mean follow-up time of 2.5 years.nnnRESULTSnThe von Willebrand factor showed a positive correlation with total mortality in the haemodialysis patients. In an unadjusted model, the hazard rate (HR) of total mortality was 2.4 [95% confidence interval (95% CI) 1.7-3.4] in the upper quartile of von Willebrand factor compared with the lowest quartile. It remained statistically significant (HR 1.8; 95% CI 1.2-2.6) after adjustment for traditional risk factors. In contrast, no significant correlation was found between von Willebrand factor levels and total mortality in PD patients. Finally, no relationship between platelet activation markers and total mortality was found in either the haemodialysis or the PD patients.nnnCONCLUSIONnIt can be concluded that chronic endothelial cell activation, but not platelet activation, is related to all-cause mortality in end-stage renal disease patients on long-term dialysis.

Chronic dysfunction of the endothelium is associated with mortality in acute coronary syndrome patients

INTRODUCTIONnPlatelet activation and endothelium dysfunction are determinants of atherothrombosis in acute coronary syndrome (ACS) patients. The aim of this study was to investigate the relationship between platelet and endothelial cell activation markers and mortality in patients presenting with ACS.nnnMATERIALS AND METHODSnPlasma levels of RANTES, Neutrophil Activating Protein-2 (NAP-2), Thrombospondin-1 (TSP-1), Von Willebrand Factor (VWF), Von Willebrand Factor Propeptide (VWF:pp) and Osteoprotegerin (OPG) were measured in a cohort study of 339 consecutive ACS patients who underwent percutaneous coronary interevention (PCI). The primary endpoint was 4-year mortality.nnnRESULTSnThere were 46 deaths during the follow up. Median values of VWF (12.2μg/mL versus 7.86μg/mL, P=0.001) and VWF:pp (7.34nM versus 6.17nM, P=0.011) were higher in non-survivors compared to survivors. High levels of OPG were found in 37 patients: 27 of them were survivors (9.2%) and 10 were non-survivors (21.7%, P=0.011). Kaplan-Meier estimates of mortality for VWF were 7.5% in the first quartile (n=6 deaths), 12.2% in the second quartile (n=10 deaths), 11.2% in the third quartile (n=9 deaths) and 25% in the fourth quartile (n=21 deaths) of VWF (P=0.004). There was a 27.8% of probability of mortality when high OPG was measured versus 12.4% when low OPG was measured (P=0.007). No relationship between baseline platelet activation markers and mortality was found.nnnCONCLUSIONnIn patients with ACS undergoing PCI, increased chronic endothelial cell activation and dysfunction is associated with an increased risk of long-term mortality.

Risk of acute ischemic heart disease in postmenopausal women depends on von Willebrand factor and fibrinogen concentrations, and blood group genotype

reagent are plotted on the x-axis, the slope of the line is known as the International Sensitivity Index (ISI). The INR is then the PTRfound in the local laboratory raised to thepowerof the ISI. This system has improved anticoagulant control worldwide. However, it is important to realize that the straight-line relationship described above is only valid for patients stably anticoagulated with VKA, and therefore this manipulation of the PTR to the INR is only valid for these patients and not for patients with other coagulation defects. For example, the INR system is not valid for comparison of patients with liver impairmentbecausedifferent reagentsdonotgive the same INR for the same sample [2]. TheHaemostasis andThrombosisTask Force of the British Committee for Standards in Haematology hasnoted thatmanyUKlaboratoriesnowreport all theirPTsas INRs.Wewish to discourage this practice, and for patientswho are not onVKA,we prefer either the PT in seconds or the PTR, in both cases with a 95% reference range for comparison. In most cases, it is prolonged results that are of interest, so it would also be acceptable to report the result with the reference being given as less than the upper limit of the 95%reference range.We accept that if both the result and the local reference range are converted to an INR, then no information is lost; however, a spurious improvement is impliedandwe suggest that this should beavoided.Converting the result toanINRwithoutprovidinga local reference range also converted to an INR is unhelpful.

Extracellular vesicles from human saliva promote hemostasis by delivering coagulant tissue factor to activated platelets

Essentials Human salivary extracellular vesicles (EVs) expose coagulant tissue factor (TF). Salivary EVs expose CD24, a ligand of P‐selectin. CD24 and coagulant TF co‐localize on salivary EVs. TF+/CD24+ salivary EVs bind to activated platelets and trigger coagulation.

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

IntroductionAt 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 methodsIn 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.ResultsIn vitro, sunitinib and sorafenib significantly inhibited platelet aggregation (20xa0μM sunitinib: 71.3%, pxa0<xa00.001; 25xa0μM sorafenib: 55.8%, pxa0=xa00.042). 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 24xa0h sunitinib impaired platelet aggregation (83.0%, pxa0=xa00.001, Nxa0=xa08). 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.ConclusionSunitinib significantly inhibits platelet aggregation in patients already after 24xa0h of first administration, whereas bevacizumab had no effect on aggregation. These findings may explain the clinically observed bleedings during treatment with antiangiogenic TKIs.

Interaction of Extracellular Vesicles with Endothelial Cells Under Physiological Flow Conditions

In the last few years it has become clear that, in addition to soluble molecules such as growth factors and cytokines, cells use extracellular vesicles (EVs) for intercellular communication. For example, EVs derived from cancer cells interact with endothelial cells, thereby affecting angiogenesis and metastasis, two essential processes in tumor progression. In most experiments, the interaction of EVs with target cells is investigated under static conditions. However the use of dynamic flow conditions is considered more relevant, especially when studying EV uptake by endothelial cells. Here, we describe the use of a perfusion system to investigate the interaction of (tumor) EVs with endothelial cells under dynamic flow conditions.