Acquired blood platelet disorder in patients with end-stage heart failure after implantation of a continuous centrifugal-flow left ventricular assist device: A prospective cohort study.

Background: Continuous-flow left ventricular assist devices (CF-LVADs) are an established therapy for advanced heart failure. Thrombosis and hemorrhage are common complications after CF-LVAD implantation, which may be explained by device-induced platelet activation. Few data on the effect of CF-LVAD implantation on platelets are available to date. Objectives: The aim of this study was to characterize the change in the platelet activation status after CF-LVAD. Methods: Platelet phenotype and reactivity were determined with flow cytometry in 32 adults with end-stage heart failure before and 4 to 6 weeks after CF-LVAD implantation. Sixteen adults with a biological aortic valve prosthesis (AVP) using the same antiplatelet regimen were included to discriminate between the effects of CF-LVAD and the antiplatelet regimen. Plasma markers for platelet activation were determined with enzyme-linked immunosorbent assay. Results: Median (IQR) plasma levels of soluble P-selectin increased from 115.6 (79.1-142.7) ng/mL to 144.5 (100.4-197.5) ng/mL after CF-LVAD implantation (P < .001). Median (IQR) ß-thromboglobulin levels were 60.5 (37.8-81.5) ng/mL before implantation and remained high after LVAD implantation [60.0 (42.0-69.5) ng/mL]. The platelet P-selectin expression after stimulation with ADP (30 and 60 µM) or PAR1-activating peptide (12.5 and 25 µM) was reduced by 17% to 21%, and fibrinogen binding was reduced by 37% to 86%. Platelet responses to agonists were similar in patients with a CF-LVAD and patients with an AVP, except for fibrinogen binding in response to 12.5 µM PAR1-AP, which was lower in patients with a CF-LVAD (P < .001). Conclusions: Combined, these data provide evidence for systemic platelet activation and an acquired platelet disorder after CF-LVAD implantation. This might contribute to the risk of both hemorrhage and thrombosis associated with CF-LVADs.

Insights in the Prothrombotic Changes After Implantation of a Left Ventricular Assist Device in Patients With End-Stage Heart Failure: A Longitudinal Observational Study.

Thrombus formation is a common complication during left ventricular assist device (LVAD) therapy, despite anticoagulation with vitamin K antagonists (VKA) and a platelet inhibitor. Plasma levels of markers for primary and secondary hemostasis and contact activation were determined before LVAD implantation and 6 and 12 months thereafter in 37 adults with end-stage heart failure. Twelve patients received a HeartMate 3, 7 patients received a HeartWare, and 18 patients received a HeartMate II. At baseline, patients had elevated plasma levels of the platelet protein upon activation, ß-thromboglobulin, and active von Willebrand factor in thrombogenic state (VWFa), which remained high after LVAD implantation. Von Willebrand factor levels and VWF activity were elevated at baseline but normalized 12 months after LVAD implantation. High D -dimer plasma levels, at baseline, remained elevated after 12 months. This was associated with an increase in plasma thrombin-antithrombin-complex levels and plasma levels of contact activation marker-cleaved H-kininogen after LVAD implantation. Considering these results it could be concluded that LVAD patients show significant coagulation activation despite antithrombotic therapy, which could explain why patients are at high risk for LVAD-induced thrombosis. Continuous low-grade systemic platelet activation and contact activation may contribute to prothrombotic effects of LVAD.

Microlyse: a thrombolytic agent that targets VWF for clearance of microvascular thrombosis.

Thrombotic microangiopathies are hallmarked by attacks of disseminated microvascular thrombosis. In thrombotic thrombocytopenic purpura (TTP), this is caused by a rise in thrombogenic ultra-large von Willebrand factor (VWF) multimers because of ADAMTS13 deficiency. We previously reported that systemic plasminogen activation is therapeutic in a TTP mouse model. In contrast to its natural activators (ie, tissue plasminogen activator and urokinase plasminogen activator [uPA]), plasminogen can directly bind to VWF. For optimal efficacy and safety, we aimed to focus and accelerate plasminogen activation at sites of microvascular occlusion. We here describe the development and characterization of Microlyse, a fusion protein consisting of a high-affinity VHH targeting the CT/CK domain of VWF and the protease domain of uPA, for localized plasminogen activation on microthrombi. Microlyse triggers targeted destruction of platelet-VWF complexes by plasmin on activated endothelial cells and in agglutination studies. At equal molar concentrations, Microlyse degrades microthrombi sevenfold more rapidly than blockade of platelet-VWF interactions with a bivalent humanized VHH (caplacizumab*). Finally, Microlyse attenuates thrombocytopenia and tissue damage (reflected by increased plasma lactate dehydrogenase activity, as well as PAI-1 and fibrinogen levels) more efficiently than caplacizumab* in an ADAMTS13-/- mouse model of TTP, without affecting hemostasis in a tail-clip bleeding model. These findings show that targeted thrombolysis of VWF by Microlyse is an effective strategy for the treatment of TTP and might hold value for other forms of VWF-driven thrombotic disease.

Osteoprotegerin modulates platelet adhesion to von Willebrand factor during release from endothelial cells.

BACKGROUND: Platelet-binding Von Willebrand Factor (VWF) strings assemble upon stimulated secretion from endothelial cells. OBJECTIVES: To investigate the efficiency of platelet binding to multi-molecular VWF bundles secreted from endothelial cells and to investigate the role of osteoprotegerin, a protein located in Weibel-Palade bodies that interacts with the VWF platelet binding domain. METHODS: The nanobody VWF/AU-a11 that specifically binds to VWF in its active platelet-binding conformation was used to investigate the conformation of VWF. RESULTS: Upon stimulated secretion from endothelial cells, VWF strings were only partially covered with platelets, while a VWD-type 2B mutation or ristocetin enhanced platelet binding by 2-3-fold. Osteoprotegrin, reduces platelet adhesion to VWF by 40% ± 18% in perfusion assays. siRNA-mediated down-regulation of endothelial osteoprotegerin expression resulted in a 1.8-fold increase in platelet adhesion to VWF strings. Upon viral infection, there is a concordant rise in VWF and osteoprotegerin plasma levels. Unexpectedly, no such increase was observed in plasma of desmopressin-treated hemophilia A-patients. In a mouse model, osteoprotegerin expression was low in liver endothelial cells of vehicle-treated mice, and concanavalin A-treatment increased VWF and osteoprotegerin expression 4- and 40-fold, respectively. This increase was translated in a 30-fold increased osteoprotegerin/VWF ratio in plasma. CONCLUSIONS: Release of VWF from endothelial cells opens the platelet-binding site, irrespective of the presence of flow. However, not all available platelet-binding sites are being occupied, suggesting some extent of regulation. Part of this regulation involves endothelial proteins that are co-secreted with VWF, like osteoprotegerin. This regulatory mechanism may be of more relevance under inflammatory conditions.

Thrombotic Events in COVID-19 Are Associated With a Lower Use of Prophylactic Anticoagulation Before Hospitalization and Followed by Decreases in Platelet Reactivity.

Background: Coronavirus disease of 2019 (COVID-19) is associated with a prothrombotic state and a high incidence of thrombotic event(s) (TE). Objectives: To study platelet reactivity in hospitalized COVID-19 patients and determine a possible association with the clinical outcomes thrombosis and all-cause mortality. Methods: Seventy nine hospitalized COVID-19 patients were enrolled in this retrospective cohort study and provided blood samples in which platelet reactivity in response to stimulation with ADP and TRAP-6 was determined using flow cytometry. Clinical outcomes included thrombotic events, and all-cause mortality. Results: The incidence of TE in this study was 28% and all-cause mortality 16%. Patients that developed a TE were younger than patients that did not develop a TE [median age of 55 vs. 70 years; adjusted odds ratio (AOR) = 0.96 per 1 year of age, 95% confidence interval (CI) 0.92-1.00; p = 0.041]. Furthermore, patients using preexisting thromboprophylaxis were less likely to develop a thrombotic complication than patients that were not (18 vs. 54%; AOR = 0.19, 95% CI 0.04-0.84; p = 0.029). Conversely, having asthma strongly increased the risk on TE development (AOR = 6.2, 95% CI 1.15-33.7; p = 0.034). No significant differences in baseline P-selectin expression or platelet reactivity were observed between the COVID-19 positive patients (n = 79) and COVID-19 negative hospitalized control patients (n = 21), nor between COVID-19 positive survivors or non-survivors. However, patients showed decreased platelet reactivity in response to TRAP-6 following TE development. Conclusion: We observed an association between the use of preexisting thromboprophylaxis and a decreased risk of TE during COVID-19. This suggests that these therapies are beneficial for coping with COVID-19 associated hypercoagulability. This highlights the importance of patient therapy adherence. We observed lowered platelet reactivity after the development of TE, which might be attributed to platelet desensitization during thromboinflammation.

Anti-ß2-glycoprotein I and anti-prothrombin antibodies cause lupus anticoagulant through different mechanisms of action.

BACKGROUND: The presence of lupus anticoagulant (LA) is an independent risk factor for thrombosis. This laboratory phenomenon is detected as a phospholipid-dependent prolongation of the clotting time and is caused by autoantibodies against beta2-glycoprotein I (ß2GPI) or prothrombin. How these autoantibodies cause LA is unclear. OBJECTIVE: To elucidate how anti-ß2GPI and anti-prothrombin antibodies cause the LA phenomenon. METHODS: The effects of monoclonal anti-ß2GPI and anti-prothrombin antibodies on coagulation were analyzed in plasma and with purified coagulation factors. RESULTS: Detection of LA caused by anti-ß2GPI or anti-prothrombin antibodies required the presence of the procofactor factor V (FV) in plasma. LA effect disappeared when FV was replaced by activated FV (FVa), both in a model system and in patient plasma, although differences between anti-ß2GPI and anti-prothrombin antibodies were observed. Further exploration of the effects of the antibodies on coagulation showed that the anti-ß2GPI antibody attenuated FV activation by activated faxtor X (FXa), whereas the anti-prothrombin antibody did not. Binding studies showed that ß2GPI--antibody complexes directly interacted with FV with high affinity. Anti-prothrombin complexes caused the LA phenomenon through competition for phospholipid binding sites with coagulation factors as reduced FXa binding to lipospheres was observed with flow cytometry in the presence of these antibodies. CONCLUSION: Anti-ß2GPI and anti-prothrombin antibodies cause LA through different mechanisms of action: While anti-ß2GPI antibodies interfere with FV activation by FXa through a direct interaction with FV, anti-prothrombin antibodies compete with FXa for phospholipid binding sites. These data provide leads for understanding the paradoxical association between thrombosis and a prolonged clotting time in the antiphospholipid syndrome.

Secondary thrombotic microangiopathy with severely reduced ADAMTS13 activity in a patient with Capnocytophaga canimorsus sepsis: a case report.

BACKGROUND: The Gram-negative bacillus Capnocytophaga canimorsus may cause a severe illness resembling thrombotic thrombocytopenic purpura (TTP). The pathogenesis and optimal therapy of this secondary thrombotic microangiopathy (TMA) remain uncertain. CASE REPORT: A 63-year-old Caucasian man was admitted with suspicion for TTP, but blood cultures grew C. canimorsus. Initial investigations revealed severe thrombocytopenia, a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) activity level of less than 1%, and strongly elevated D-dimer and lactate dehydrogenase levels. He made a full recovery with antibiotics and plasma infusion for 3 days. Plasmapheresis was not performed. Retrospective determination of serial ADAMTS13 activity levels revealed that ADAMTS13 activity had already increased to 25% at the start of plasma infusion. CONCLUSION: This case highlights that a C. canimorsus sepsis may cause a secondary TMA with a severe ADAMTS13 deficiency. It also illustrates that the adjunctive role of plasma exchange or plasma infusion is doubtful as ADAMTS13 activity levels increased with antibiotics alone.

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.

The functions of the A1A2A3 domains in von Willebrand factor include multimerin 1 binding.

Multimerin 1 (MMRN1) is a massive, homopolymeric protein that is stored in platelets and endothelial cells for activation-induced release. In vitro, MMRN1 binds to the outer surfaces of activated platelets and endothelial cells, the extracellular matrix (including collagen) and von Willebrand factor (VWF) to support platelet adhesive functions. VWF associates with MMRN1 at high shear, not static conditions, suggesting that shear exposes cryptic sites within VWF that support MMRN1 binding. Modified ELISA and surface plasmon resonance were used to study the structural features of VWF that support MMRN1 binding, and determine the affinities for VWF-MMRN1 binding. High shear microfluidic platelet adhesion assays determined the functional consequences for VWF-MMRN1 binding. VWF binding to MMRN1 was enhanced by shear exposure and ristocetin, and required VWF A1A2A3 region, specifically the A1 and A3 domains. VWF A1A2A3 bound to MMRN1 with a physiologically relevant binding affinity (KD: 2.0 ± 0.4 nM), whereas the individual VWF A1 (KD: 39.3 ± 7.7 nM) and A3 domains (KD: 229 ± 114 nM) bound to MMRN1 with lower affinities. VWF A1A2A3 was also sufficient to support the adhesion of resting platelets to MMRN1 at high shear, by a mechanism dependent on VWF-GPIbα binding. Our study provides new information on the molecular basis of MMRN1 binding to VWF, and its role in supporting platelet adhesion at high shear. We propose that at sites of vessel injury, MMRN1 that is released following activation of platelets and endothelial cells, binds to VWF A1A2A3 region to support platelet adhesion at arterial shear rates.

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.

Imbalance of angiopoietin-1 and angiopoetin-2 in severe dengue and relationship with thrombocytopenia, endothelial activation, and vascular stability.

The pathogenesis of plasma leakage during dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) is largely unknown. Angiopoietins are key regulators of vascular integrity: Angiopoietin-1 is stored in platelets and maintains vascular integrity, and endothelium-derived angiopoietin-2 promotes vascular leakage. We determined angiopoietin-1 and angiopoietin-2 levels in a cohort of children in Indonesia with DHF/DSS and related them to plasma leakage markers. Patients with DHF/DSS had reduced angiopoietin-1 and increased angiopoietin-2 plasma levels on the day of admission when compared with levels at discharge and in healthy controls. There was an inverse correlation between angiopoietin-1 and markers of plasma leakage and a positive correlation between angiopoietin-2 and markers of plasma leakage. Angiopoietin-1 levels followed the same trend as the soluble platelet activation marker P-selectin and correlated with platelet counts. Dengue-associated thrombocytopenia and endothelial activation are associated with an imbalance in angiopoietin-2: angiopoietin-1 plasma levels. This imbalance may contribute to the transient plasma leakage in DHF/DSS.

Severe dengue is associated with consumption of von Willebrand factor and its cleaving enzyme ADAMTS-13.

BACKGROUND: Thrombocytopenia, bleeding and plasma leakage are cardinal features of severe dengue. Endothelial cell activation with exocytosis of Weibel-Palade bodies (WPBs) may play an etiological role in this condition. METHODS AND PRINCIPAL FINDINGS: In a cohort of 73 Indonesian children with dengue hemorrhagic fever (DHF), of which 30 with dengue shock syndrome (DSS), we measured plasma levels of the WPB constituents von Willebrand factor antigen (VWF:Ag), VWF propeptide and osteoprotegerin (OPG), together with activity levels of the VWF-cleaving enzyme ADAMTS-13 and the amount of VWF in a platelet binding conformation (VWF activation factor). Compared with healthy controls (n = 17), children with DHF/DSS had significantly higher levels of VWF:Ag, VWF propeptide and OPG and decreased ADAMTS-13 activity. The VWF activation factor was also significantly higher in DHF/DSS and highest in children who died. There were significant differences in the kinetics of the various WPB constituents: VWF propeptide and OPG levels decreased toward discharge, while VWF:Ag levels were lower than expected at enrollment with plasma levels increasing toward discharge. Moreover, VWF propeptide levels correlated better with markers of disease severity (platelet count, liver enzymes, serum albumin and pleural effusion index) than corresponding VWF levels. Together, these findings suggest that there is consumption of VWF in DHF/DSS. In 4 out of 15 selected children with low ADAMTS-13 levels on admission, we found a remarkable reduction in the large and intermediate VWF multimers in the discharge blood samples, consistent with an acquired von Willebrand disease. CONCLUSION: These findings suggest that severe dengue is associated with exocytosis of WPBs with increased circulating levels of VWF:Ag, VWF propeptide and OPG. High circulating levels of VWF in its active conformation, together with low ADAMTS-13 activity levels, are likely to contribute to the thrombocytopenia and complications of dengue. During the convalescence phase, qualitative defects in VWF with loss of larger VWF multimers may develop.

The Staphylococcus aureus lineage-specific markers collagen adhesin and toxic shock syndrome toxin 1 distinguish multilocus sequence typing clonal complexes within spa clonal complexes.

Spa typing/based upon repeat pattern (BURP) sometimes cannot differentiate multilocus sequence typing (MLST) clonal complexes (CCs) within spa-CCs. It has been observed previously that virulence factors, such as collagen adhesin (CNA) and toxic shock syndrome toxin 1 (TSST-1), are associated with certain Staphylococcus aureus lineages. Analysis of methicillin-sensitive and methicillin-resistant S. aureus by spa typing/BURP and detection of CNA and TSST-1 observed an association between CNA and MLST CC1, 12, 22, 30, 45, 51, and 239 and between TSST-1 and MLST CC30. In spa-CC 012, associated with MLST CC7, CC15, and CC30, MLST CC30 could be distinguished from MLST CC7 and CC15 with CNA and TSST-1 as lineage-specific markers. Lineage-specific markers can overcome clustering of nonrelated MLST CCs into 1 spa-CC.

Cross-border dissemination of methicillin-resistant Staphylococcus aureus, Euregio Meuse-Rhin region.

Because the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) differs among the 3 countries forming the Euregio Meuse-Rhin (EMR) region (Belgium, Germany, and the Netherlands), cross-border healthcare requires information about the spread of MRSA in the EMR. We investigated the emergence, dissemination, and diversity of MRSA clones in the EMR by using several typing methods. MRSA associated with clonal complexes 5, 8, 30, and 45 was disseminated throughout the EMR. Dutch isolates, mainly associated with sequence types (ST) ST5-MRSA-II, ST5-MRSA-IV, ST8-MRSA-IV, and ST45-MSRA-IV had a more diverse genetic background than the isolates from Belgium and Germany, associated with ST45-MRSA-IV and ST5-MRSA-II, respectively. MRSA associated with pigs (ST398-MRSA-IV/V) was found in the Dutch area of the EMR. Five percent of the MRSA isolates harbored Panton-Valentine leukocidin and were classified as community-associated MRSA associated with ST1, 8, 30, 80, and 89.

ADAMTS13 deficiency with elevated levels of ultra-large and active von Willebrand factor in P. falciparum and P. vivax malaria.

A deficiency in ADAMTS13 (a von Willebrand factor [VWF] cleaving protease) is associated with accumulation of prothrombogenic unusually large VWF multimers (UL-VWF) in plasma. We studied VWF release and proteolysis in patients with symptomatic Plasmodium falciparum or P. vivax malaria on the Indonesian island Sumba. Malaria patients had significantly lower platelet counts and higher VWF concentrations and VWF activation factors than healthy hospital staff controls. The latter indicates that a higher amount of circulating VWF was in a conformation enabling spontaneous platelet binding. In addition, ADAMTS13 activity and antigen levels were reduced in both malaria groups, and this was associated with the appearance of UL-VWF. The mechanism behind this reduction and the role in malaria pathogenesis needs to be further elucidated. In malaria, endothelial cell activation with increased circulating amounts of active and ultra-large VWF, together with reduced VWF inactivation by ADAMTS13, may result in intravascular platelet aggregation, thrombocytopenia, and microvascular disease.

Molecular characterization of Staphylococcus aureus bloodstream isolates collected in a Dutch University Hospital between 1999 and 2006.

We observed that, between 1999 and 2006, up to 50% of the methicillin-susceptible Staphylococcus aureus (MSSA) bloodstream isolates in our hospital had a genetic background common to endemic methicillin-resistant S. aureus clones (clonal complex 5 [CC5], CC8, CC22, CC30, and CC45). Furthermore, several successful MSSA lineages, such as CC7 and CC15, were observed.