Design, manufacturing and testing of a green non-isocyanate polyurethane prosthetic heart valve.

The sole effective treatment for most patients with heart valve disease is valve replacement by implantation of mechanical or biological prostheses. However, mechanical valves represent high risk of thromboembolism, and biological prostheses are prone to early degeneration. In this work, we aim to determine the potential of novel environmentally-friendly non-isocyanate polyurethanes (NIPUs) for manufacturing synthetic prosthetic heart valves. Polyhydroxyurethane (PHU) NIPUs are synthesized via an isocyanate-free route, tested in vitro, and used to produce aortic valves. PHU elastomers reinforced with a polyester mesh show mechanical properties similar to native valve leaflets. These NIPUs do not cause hemolysis. Interestingly, both platelet adhesion and contact activation-induced coagulation are strongly reduced on NIPU surfaces, indicating low thrombogenicity. Fibroblasts and endothelial cells maintain normal growth and shape after indirect contact with NIPUs. Fluid-structure interaction (FSI) allows modeling of the ideal valve design, with minimal shear stress on the leaflets. Injection-molded valves are tested in a pulse duplicator and show ISO-compliant hydrodynamic performance, comparable to clinically-used bioprostheses. Poly(tetrahydrofuran) (PTHF)-NIPU patches do not show any evidence of calcification over a period of 8 weeks. NIPUs are promising sustainable biomaterials for the manufacturing of improved prosthetic valves with low thrombogenicity.

Endothelium-mediated regulation of platelet activation: Involvement of multiple protein kinases.

The endothelial regulation of platelet activity is incompletely understood. Here we describe novel approaches to find molecular pathways implicated on the platelet-endothelium interaction. Using high-shear whole-blood microfluidics, employing coagulant or non-coagulant conditions at physiological temperature, we observed that the presence of human umbilical vein endothelial cells (HUVEC) strongly suppressed platelet adhesion and activation, via the collagen receptor glycoprotein VI (GPVI) and the PAR receptors for thrombin. Real-time monitoring of the cytosolic Ca2+ rises in the platelets indicated no major improvement of inhibition by prostacyclin or nitric oxide. Similarly under stasis, exposure of isolated platelets to HUVEC reduced the Ca2+ responses by collagen-related peptide (CRP-XL, GPVI agonist) and thrombin (PAR agonist). We then analyzed the label-free phosphoproteome of platelets (three donors), exposed to HUVEC, CRP-XL, and/or thrombin. High-resolution mass spectrometry gave 5463 phosphopeptides, corresponding to 1472 proteins, with good correlation between biological and technical replicates (R > .86). Stringent filtering steps revealed 26 regulatory pathways (Reactome) and 143 regulated kinase substrates (PhosphoSitePlus), giving a set of protein phosphorylation sites that was differentially (44) or similarly (110) regulated by HUVEC or agonist exposure. The differential regulation was confirmed by stable-isotope analysis of platelets from two additional donors. Substrate analysis indicated major roles of poorly studied protein kinase classes (MAPK, CDK, DYRK, STK, PKC members). Collectively, these results reveal a resetting of the protein phosphorylation profile in platelets exposed to endothelium or to conventional agonists and to endothelium-promoted activity of a multi-kinase network, beyond classical prostacyclin and nitric oxide actors, that may contribute to platelet inhibition.

Antagonistic Roles of Human Platelet Integrin αIIbß3 and Chemokines in Regulating Neutrophil Activation and Fate on Arterial Thrombi Under Flow.

BACKGROUND: Platelets and neutrophils are the first blood cells accumulating at sites of arterial thrombus formation, and both cell types contribute to the pathology of thrombotic events. We aimed to identify key interaction mechanisms between these cells using microfluidic approaches. METHODS: Whole-blood perfusion was performed over a collagen surface at arterial shear rate. Platelet and leukocyte (in majority neutrophil) activation were microscopically visualized using fluorescent markers. The contributions of platelet-adhesive receptors (integrin, P-selectin, CD40L) and chemokines were studied by using inhibitors or antibodies and using blood from patients with GT (Glanzmann thrombasthenia) lacking platelet-expressed αIIbß3. RESULTS: We observed (1) an unknown role of activated platelet integrin αIIbß3 preventing leukocyte adhesion, which was overcome by short-term flow disturbance provoking massive adhesion; (2) that platelet-expressed CD40L controls the crawling pattern and thrombus fidelity of the cells on a thrombus; (3) that continued secretion of platelet substances promotes activation of identified neutrophils, as assessed by (fMLP [N-formylmethionyl-leucyl-phenylalanine, a potent chemotactic agent and leukocyte activator] induced) [Ca2+]i rises and antigen expression; (4) and that platelet-released chemokines activate the adhered cells in the order of CXCL7>CCL5>CXCL4. Furthermore, postsilencing of the platelets in a thrombus suppressed the leukocyte activation. However, the leukocytes on thrombi did no more than limitedly form neutrophil extracellular traps, unless stimulated with phorbol ester or lipopolysaccharide. CONCLUSIONS: Together, these findings reveal a multifaceted regulation of adhesion and activation of neutrophils by platelets in a thrombus, with a balanced role of several platelet-adhesive receptors and a promoting role of platelet-released substances. This multivalent nature of neutrophil-thrombus interactions offers novel prospects for pharmacological intervention.

Unraveling the role of the homoarginine residue in antiplatelet drug eptifibatide in binding to the αIIbß3 integrin receptor.

Eptifibatide is an αIIbß3 inhibitor that is currently used in the clinic. More than 10 scientific communications indicate that eptifibatide has a Lys-Gly-Asp or Arg-Gly-Asp sequence, while it actually has a hArg-Gly-Asp sequence. We aimed to unravel the importance of the homoarginine residue in eptifibatide in platelet activation and aggregation. Arg- and Lys-eptifibatide were synthesized by solid-phase peptide synthesis and measured in light transmission aggregometry, flow cytometry and whole blood thrombus formation under flow. Interactions of eptifibatide and its variants with αIIbß3 integrin were studied using molecular dynamics simulations. Eptifibatide showed inhibition of collagen- and ADP-induced platelet aggregation, while Arg- and Lys-eptifibatide did not. Multiparameter assessment of thrombus formation showed suppressed platelet aggregate and fibrin formation upon eptifibatide treatment, in contrast to the other variants. Molecular dynamics simulations revealed that the hArg residue in eptifibatide is crucial to its activity, since the substitution of the hArg to Arg or Lys resulted in the inability to form double H-bonds with Asp224 in the αIIb chain of the αIIbß3 receptor. The hArg is pivotal for the interaction of eptifibatide for the αIIbß3 receptor and efficient inhibition of platelet aggregation.

Quantitative and qualitative changes in platelet traits of sunitinib-treated patients with renal cell carcinoma in relation to circulating sunitinib levels: a proof-of-concept study.

BACKGROUND: Tyrosine kinase inhibitors (TKIs), such as sunitinib, are used for cancer treatment, but may also affect platelet count and function with possible hemostatic consequences. Here, we investigated whether patient treatment with the TKI sunitinib affected quantitative and qualitative platelet traits as a function of the sunitinib level and the occurrence of bleeding. METHODS: Blood was collected from 20 metastatic renal cell carcinoma (mRCC) patients before treatment, and at 2 weeks, 4 weeks and 3 months after sunitinib administration. We measured blood cell counts, platelet aggregation, and concentrations of sunitinib as well as its N-desethyl metabolite in plasma, serum and isolated platelets. Progression of disease (PD) and bleeding were monitored after 3 months. RESULTS: In sunitinib-treated mRCC patients, concentrations of (N-desethyl-)sunitinib in plasma and serum were highly correlated. In the patients' platelets the active metabolite levels were relatively increased as compared to sunitinib. On average, a sustained reduction in platelet count was observed on-treatment, which was significantly related to the inhibitor levels in plasma/serum. Principal component and correlational analysis showed that the (N-desethyl-)sunitinib levels in plasma/serum were linked to a reduction in both platelet count and collagen-induced platelet aggregation. The reduced aggregation associated in part with reported bleeding, but did not correlate to PD. CONCLUSION: The sunitinib-induced reduction in quantitative and qualitative platelet traits may reflect the effective sunitinib levels in the patient. These novel results may serve as a proof-of-principle for other TKI-related drugs, where both platelet count and functions are affected, which could be used for therapeutic drug monitoring.

GPVI expression is linked to platelet size, age, and reactivity.

Platelets within one individual display heterogeneity in reactivity, size, age, and expression of surface receptors. To investigate the combined intraindividual contribution of platelet size, platelet age, and receptor expression levels on the reactivity of platelets, we studied fractions of large and small platelets from healthy donors separated by using differential centrifugation. Size-separated platelet fractions were perfused over a collagen-coated surface to assess thrombus formation. Multicolor flow cytometry was used to characterize resting and stimulated platelet subpopulations, and platelet age was determined based on RNA and HLA-I labeling. Signal transduction was analyzed by measuring consecutive phosphorylation of serine/threonine-protein kinase Akt. Compared with small platelets, large platelets adhered faster to collagen under flow and formed larger thrombi. Among the large platelets, a highly reactive juvenile platelet subpopulation was identified with high glycoprotein VI (GPVI) expression. Elevated GPVI expression correlated with high HLA-I expression, RNA content, and increased platelet reactivity. There was a stronger difference in Akt phosphorylation and activation upon collagen stimulation between juvenile and older platelets than between large and small platelets. GPVI expression and platelet reactivity decreased throughout platelet storage at 22°C and was better maintained throughout cold storage at 4°C. We further detected higher GPVI expression in platelets of patients with immune thrombocytopenia. Our findings show that high GPVI expression is a feature of highly reactive juvenile platelets, which are predominantly found among the large platelet population, explaining the better performance of large platelets during thrombus formation. These data are important for studies of thrombus formation, platelet storage, and immune thrombocytopenia.

Reversing direct factor Xa or thrombin inhibitors: Factor V addition to prothrombin complex concentrate is beneficial in vitro.

Background: Prothrombin complex concentrate (PCC) is a human plasma-derived mixture of partially purified vitamin K-dependent coagulation factors (VKCF). Current therapeutic indication is treatment and perioperative prophylaxis of bleeding in acquired VKCF deficiency. Off-label uses include treatment of direct factor Xa- or thrombin inhibitor-associated bleeds, treatment of trauma-induced coagulopathy, and hemorrhagic complications in patients with liver disease. Objective: Considering PCC as a general prohemostatic drug, we argued that its clinical efficacy can benefit from supplementation with coagulation factors that are absent in the current PCC formulation. In this study, we focused on factor V. Methods: We mimicked a coagulopathy in vitro by spiking whole blood or derived plasma with the direct oral anticoagulants (DOAC) rivaroxaban or dabigatran. We studied DOAC reversal by PCC and factor V concentrate (FVC) using a thrombin generation assay, thromboelastography, fibrin generation clot lysis test, and microfluidic thrombus formation under flow. Results: In DOAC-treated plasma, PCC increased the amount of thrombin generated. The addition of FVC alone or in combination with PCC caused a partial correction of the thrombin generation lag time and clotting time. In DOAC-treated whole blood, the combination of PCC and FVC synergistically improved clotting time under static conditions, whereas complete correction of fibrin formation was observed under flow. Clot strength and clot resistance toward tissue plasminogen activator-induced lysis were both increased with PCC and further enhanced by additional FVC. Conclusion: Our in vitro study demonstrates a beneficial effect of the combined use of PCC and FVC in DOAC reversal.

Protein C or Protein S deficiency associates with paradoxically impaired platelet-dependent thrombus and fibrin formation under flow.

Background: Low plasma levels of protein C or protein S are associated with venous thromboembolism rather than myocardial infarction. The high coagulant activity in patients with thrombophilia with a (familial) defect in protein C or S is explained by defective protein C activation, involving thrombomodulin and protein S. This causes increased plasmatic thrombin generation. Objective: Assess the role of platelets in the thrombus- and fibrin-forming potential in patients with familial protein C or protein S deficiency under high-shear flow conditions. Patients/Methods: Whole blood from 23 patients and 15 control subjects was perfused over six glycoprotein VI-dependent microspot surfaces. By real-time multicolor microscopic imaging, kinetics of platelet thrombus and fibrin formation were characterized in 49 parameters. Results and Conclusion: Whole-blood flow perfusion over collagen, collagen-like peptide, and fibrin surfaces with low or high GPVI dependency indicated an unexpected impairment of platelet activation, thrombus phenotype, and fibrin formation but unchanged platelet adhesion, observed in patients with protein C deficiency and to a lesser extent protein S deficiency, when compared to controls. The defect extended from diminished phosphatidylserine exposure and thrombus contraction to delayed and suppressed fibrin formation. The mechanism was thrombomodulin independent, and may involve negative platelet priming by plasma components.

Multiparameter platelet function analysis of bleeding patients with a prolonged platelet function analyser closure time.

Patients referred for evaluation of bleeding symptoms occasionally have a prolonged platelet function analyser (PFA) closure time, without evidence for von Willebrand disease or impaired platelet aggregation. The aim of this study was to establish a shear-dependent platelet function defect in these patients. Patients were included based on high bleeding score and prior PFA prolongation. Common tests of von Willebrand factor (VWF) and platelet function and exome sequencing were performed. Microfluidic analysis of shear-dependent collagen-induced whole-blood thrombus formation was performed. In 14 PFA-only patients, compared to healthy volunteers, microfluidic tests showed significantly lower platelet adhesion and thrombus formation parameters. This was accompanied by lower integrin activation, phosphatidylserine exposure and P-selectin expression. Principal components analysis indicated VWF as primary explaining variable of PFA prolongation, whereas conventional platelet aggregation primarily explained the reduced thrombus parameters under shear. In five patients with severe microfluidic abnormalities, conventional platelet aggregation was in the lowest range of normal. No causal variants in Mendelian genes known to cause bleeding or platelet disorders were identified. Multiparameter assessment of whole-blood thrombus formation under shear indicates single or combined effects of low-normal VWF and low-normal platelet aggregation in these patients, suggesting a shear-dependent platelet function defect, not detected by static conventional haemostatic tests.

Rapid Internalization and Nuclear Translocation of CCL5 and CXCL4 in Endothelial Cells.

The chemokines CCL5 and CXCL4 are deposited by platelets onto endothelial cells, inducing monocyte arrest. Here, the fate of CCL5 and CXCL4 after endothelial deposition was investigated. Human umbilical vein endothelial cells (HUVECs) and EA.hy926 cells were incubated with CCL5 or CXCL4 for up to 120 min, and chemokine uptake was analyzed by microscopy and by ELISA. Intracellular calcium signaling was visualized upon chemokine treatment, and monocyte arrest was evaluated under laminar flow. Whereas CXCL4 remained partly on the cell surface, all of the CCL5 was internalized into endothelial cells. Endocytosis of CCL5 and CXCL4 was shown as a rapid and active process that primarily depended on dynamin, clathrin, and G protein-coupled receptors (GPCRs), but not on surface proteoglycans. Intracellular calcium signals were increased after chemokine treatment. Confocal microscopy and ELISA measurements in cell organelle fractions indicated that both chemokines accumulated in the nucleus. Internalization did not affect leukocyte arrest, as pretreatment of chemokines and subsequent washing did not alter monocyte adhesion to endothelial cells. Endothelial cells rapidly and actively internalize CCL5 and CXCL4 by clathrin and dynamin-dependent endocytosis, where the chemokines appear to be directed to the nucleus. These findings expand our knowledge of how chemokines attract leukocytes to sites of inflammation.

Cell-specific and divergent roles of the CD40L-CD40 axis in atherosclerotic vascular disease.

Atherosclerosis is a major underlying cause of cardiovascular disease. Previous studies showed that inhibition of the co-stimulatory CD40 ligand (CD40L)-CD40 signaling axis profoundly attenuates atherosclerosis. As CD40L exerts multiple functions depending on the cell-cell interactions involved, we sought to investigate the function of the most relevant CD40L-expressing cell types in atherosclerosis: T cells and platelets. Atherosclerosis-prone mice with a CD40L-deficiency in CD4+ T cells display impaired Th1 polarization, as reflected by reduced interferon-γ production, and smaller atherosclerotic plaques containing fewer T-cells, smaller necrotic cores, an increased number of smooth muscle cells and thicker fibrous caps. Mice with a corresponding CD40-deficiency in CD11c+ dendritic cells phenocopy these findings, suggesting that the T cell-dendritic cell CD40L-CD40 axis is crucial in atherogenesis. Accordingly, sCD40L/sCD40 and interferon-γ concentrations in carotid plaques and plasma are positively correlated in patients with cerebrovascular disease. Platelet-specific deficiency of CD40L does not affect atherogenesis but ameliorates atherothrombosis. Our results establish divergent and cell-specific roles of CD40L-CD40 in atherosclerosis, which has implications for therapeutic strategies targeting this pathway.

Inhibition of platelet adhesion, thrombus formation, and fibrin formation by a potent αIIbß3 integrin inhibitor from ticks.

BACKGROUND: Ticks puncture the skin of their hosts and secrete saliva, containing antiplatelet proteins, into the blood. Here, we studied disagregin, a potent platelet-inhibiting protein derived from the salivary glands of Ornithodoros moubata, an African soft tick. Whereas conventional αIIbß3 antagonists contain an Arg-Gly-Asp (RGD) sequence for platelet integrin binding, disagregin contains an Arg-Glu-Asp (RED) sequence, hypothesizing a different mode of inhibitory action. OBJECTIVES: We aimed to compare the inhibitory effects of disagregin and its RGD variant (RGD-disagregin) on platelet activation and to unravel the molecular basis of disagregin-αIIbß3 integrin interactions. METHODS: Disagregin and RGD-disagregin were synthesized by tert-butyloxycarbonyl -based solid-phase peptide synthesis. Effects of both disagregins on platelet aggregation were assessed by light transmission aggregometry in human platelet-rich plasma. Whole-blood thrombus formation was investigated by perfusing blood over collagen I with and without tissue factor at a high wall-shear rate (1000 s-1) in the presence of disagregin, RGD-disagregin, or eptifibatide. RESULTS: Disagregin showed inhibition of collagen- and ADP-induced platelet aggregation with half maximal inhibitory concentration values of 64 and 99 nM, respectively. This resembled the complete antiaggregatory effect of eptifibatide. Multiparameter assessment of thrombus formation showed highly suppressed platelet adhesion and aggregate formation with both disagregins, in contrast to eptifibatide treatment, which incompletely blocked aggregation under flow. Fibrin formation under flow was delayed by both disagregin and RGD-disagregin (P < .01) and eptifibatide (P < .05). CONCLUSIONS: Both αIIbß3-blocking disagregins have a strong potential to suppress collagen-tissue factor-mediated platelet adhesion, thrombus formation, and fibrin formation. Both disagregins can be seen as potential new αIIbß3 inhibitors.

ADAM10-Mediated Cleavage of ICAM-1 Is Involved in Neutrophil Transendothelial Migration.

To efficiently cross the endothelial barrier during inflammation, neutrophils first firmly adhere to the endothelial surface using the endothelial adhesion molecule ICAM-1. Upon actual transmigration, the release from ICAM-1 is required. While Integrin LFA1/Mac1 de-activation is one described mechanism that leads to this, direct cleavage of ICAM-1 from the endothelium represents a second option. We found that a disintegrin and metalloprotease 10 (ADAM10) cleaves the extracellular domain of ICAM-1 from the endothelial surface. Silencing or inhibiting endothelial ADAM10 impaired the efficiency of neutrophils to cross the endothelium, suggesting that neutrophils use endothelial ADAM10 to dissociate from ICAM-1. Indeed, when measuring transmigration kinetics, neutrophils took almost twice as much time to finish the diapedesis step when ADAM10 was silenced. Importantly, we found increased levels of ICAM-1 on the transmigrating neutrophils when crossing an endothelial monolayer where such increased levels were not detected when neutrophils crossed bare filters. Using ICAM-1-GFP-expressing endothelial cells, we show that ICAM-1 presence on the neutrophils can also occur by membrane transfer from the endothelium to the neutrophil. Based on these findings, we conclude that endothelial ADAM10 contributes in part to neutrophil transendothelial migration by cleaving ICAM-1, thereby supporting the release of neutrophils from the endothelium during the final diapedesis step.

Platelet-primed interactions of coagulation and anticoagulation pathways in flow-dependent thrombus formation.

In haemostasis and thrombosis, platelet, coagulation and anticoagulation pathways act together to produce fibrin-containing thrombi. We developed a microspot-based technique, in which we assessed platelet adhesion, platelet activation, thrombus structure and fibrin clot formation in real time using flowing whole blood. Microspots were made from distinct platelet-adhesive surfaces in the absence or presence of tissue factor, thrombomodulin or activated protein C. Kinetics of platelet activation, thrombus structure and fibrin formation were assessed by fluorescence microscopy. This work revealed: (1) a priming role of platelet adhesion in thrombus contraction and subsequent fibrin formation; (2) a surface-independent role of tissue factor, independent of the shear rate; (3) a mechanism of tissue factor-enhanced activation of the intrinsic coagulation pathway; (4) a local, suppressive role of the anticoagulant thrombomodulin/protein C pathway under flow. Multiparameter analysis using blood samples from patients with (anti)coagulation disorders indicated characteristic defects in thrombus formation, in cases of factor V, XI or XII deficiency; and in contrast, thrombogenic effects in patients with factor V-Leiden. Taken together, this integrative phenotyping approach of platelet-fibrin thrombus formation has revealed interaction mechanisms of platelet-primed key haemostatic pathways with alterations in patients with (anti)coagulation defects. It can help as an important functional add-on whole-blood phenotyping.

Crystal Clots as Therapeutic Target in Cholesterol Crystal Embolism.

RATIONALE: Cholesterol crystal embolism can be a life-threatening complication of advanced atherosclerosis. Pathophysiology and molecular targets for treatment are largely unknown. OBJECTIVE: We aimed to develop a new animal model of cholesterol crystal embolism to dissect the molecular mechanisms of cholesterol crystal (CC)-driven arterial occlusion, tissue infarction, and organ failure. METHODS AND RESULTS: C57BL/6J mice were injected with CC into the left kidney artery. Primary end point was glomerular filtration rate (GFR). CC caused crystal clots occluding intrarenal arteries and a dose-dependent drop in GFR, followed by GFR recovery within 4 weeks, that is, acute kidney disease. In contrast, the extent of kidney infarction was more variable. Blocking necroptosis using mixed lineage kinase domain-like deficient mice or necrostatin-1s treatment protected from kidney infarction but not from GFR loss because arterial obstructions persisted, identifying crystal clots as a primary target to prevent organ failure. CC involved platelets, neutrophils, fibrin, and extracellular DNA. Neutrophil depletion or inhibition of the release of neutrophil extracellular traps had little effects, but platelet P2Y12 receptor antagonism with clopidogrel, fibrinolysis with urokinase, or DNA digestion with recombinant DNase I all prevented arterial occlusions, GFR loss, and kidney infarction. The window-of-opportunity was <3 hours after CC injection. However, combining Nec-1s (necrostatin-1s) prophylaxis given 1 hour before and DNase I 3 hours after CC injection completely prevented kidney failure and infarcts. In vitro, CC did not directly induce plasmatic coagulation but induced neutrophil extracellular trap formation and DNA release mainly from kidney endothelial cells, neutrophils, and few from platelets. CC induced ATP release from aggregating platelets, which increased fibrin formation in a DNase-dependent manner. CONCLUSIONS: CC embolism causes arterial obstructions and organ failure via the formation of crystal clots with fibrin, platelets, and extracellular DNA as critical components. Therefore, our model enables to unravel the pathogenesis of the CC embolism syndrome as a basis for both prophylaxis and targeted therapy.

Localized endothelial-based control of platelet aggregation and coagulation under flow: A proof-of-principle vessel-on-a-chip study.

BACKGROUND: In the intact vessel wall, endothelial cells form a barrier between the blood and the remaining vascular structures, serving to maintain blood fluidity and preventing platelet activation and fibrin clot formation. The spatiotemporal space of this inhibition is largely unknown. OBJECTIVE: To assess the local inhibitory roles of a discontinuous endothelium, we developed a vessel-on-a-chip model, consisting of a microfluidic chamber coated with the thrombogenic collagen and tissue factor (TF), and covered with patches of human endothelial cells. By flow perfusion of human blood and plasma, the heterogeneous formation of platelet aggregates and fibrin clots was monitored by multicolor fluorescence microscopy. RESULTS: On collagen/TF coatings, a coverage of 40% to 60% of human umbilical vein endothelial cells resulted in a strong overall delay in platelet deposition and fibrin fiber formation under flow. Fibrin formation colocalized with the deposited platelets, and was restricted to regions in between endothelial cells, thus pointing to immediate local suppression of the clotting process. Fibrin kinetics were enhanced by treatment of the cells with heparinase III, partially disrupting the glycocalyx, and to a lesser degree by antagonism of the endothelial thrombomodulin. Co-coating of purified thrombomodulin and collagen had a similar coagulation-suppressing effect as endothelial thrombomodulin. CONCLUSIONS: In this vessel-on-a-chip system with patches of endothelial cells on thrombogenic surfaces, the coagulant activity under flow is regulated by: (a) the residual exposure of trigger (collagen/TF), (b) the endothelial glycocalyx, and (c) to a lesser degree the endothelial thrombomodulin.

Whole Blood Based Multiparameter Assessment of Thrombus Formation in Standard Microfluidic Devices to Proxy In Vivo Haemostasis and Thrombosis.

Microfluidic assays are versatile tests which, using only small amounts of blood, enable high throughput analyses of platelet function in several minutes. In combination with fluorescence microscopy, these flow tests allow real-time visualisation of platelet activation with the possibility of examining combinatorial effects of wall shear rate, coagulation and modulation by endothelial cells. In particular, the ability to use blood and blood cells from healthy subjects or patients makes this technology promising, both for research and (pre)clinical diagnostic purposes. In the present review, we describe how microfluidic devices are used to assess the roles of platelets in thrombosis and haemostasis. We place emphasis on technical aspects and on experimental designs that make the concept of "blood-vessel-component-on-a-chip" an attractive, rapidly developing technology for the study of the complex biological processes of blood coagulability in the presence of flow.

Clinical Protocol to Prevent Thrombogenic Effect of Liver-Derived Mesenchymal Cells for Cell-Based Therapies.

The efficacy of mesenchymal stem cell infusion is currently tested in numerous clinical trials. However, therapy-induced thrombotic consequences have been reported in several patients. The aim of this study was to optimize protocols for heterologous human adult liver-derived progenitor cell (HHALPC) infusion, in order to eliminate acute thrombogenesis in liver-based metabolic or acute decompensated cirrhotic (ADC) patients. In rats, thrombotic effects were absent when HHALPCs were infused at low cell dose (5 × 106 cells/kg), or at high cell dose (5 × 107 cells/kg) when combined with anticoagulants. When HHALPCs were exposed to human blood in a whole blood perfusion assay, blocking of the tissue factor (TF) coagulation pathway suppressed fibrin generation and platelet activation. In a Chandler tubing loop model, HHALPCs induced less explosive activation of coagulation with blood from ADC patients, when compared to blood from healthy controls, without alterations in coagulation factor levels other than fibrinogen. These studies confirm a link between TF and thrombogenesis, when TF-expressing cells are exposed to human blood. This phenomenon however, could be controlled using either a low, or a high cell dose combined with anticoagulants. In clinical practice, this points to the suitability of a low HHALPC dose infusion to cirrhotic patients, provided that platelet and fibrinogen levels are monitored.

Defective Zn2+ homeostasis in mouse and human platelets with α- and δ-storage pool diseases.

Zinc (Zn2+) can modulate platelet and coagulation activation pathways, including fibrin formation. Here, we studied the (patho)physiological consequences of abnormal platelet Zn2+ storage and release. To visualize Zn2+ storage in human and mouse platelets, the Zn2+ specific fluorescent dye FluoZin3 was used. In resting platelets, the dye transiently accumulated into distinct cytosolic puncta, which were lost upon platelet activation. Platelets isolated from Unc13d-/- mice, characterized by combined defects of α/δ granular release, showed a markedly impaired Zn2+ release upon activation. Platelets from Nbeal2-/- mice mimicking Gray platelet syndrome (GPS), characterized by primarily loss of the α-granule content, had strongly reduced Zn2+ levels, which was also confirmed in primary megakaryocytes. In human platelets isolated from patients with GPS, Hermansky-Pudlak Syndrome (HPS) and Storage Pool Disease (SPD) altered Zn2+ homeostasis was detected. In turbidity and flow based assays, platelet-dependent fibrin formation was impaired in both Nbeal2-/- and Unc13d-/- mice, and the impairment could be partially restored by extracellular Zn2+. Altogether, we conclude that the release of ionic Zn2+ store from secretory granules upon platelet activation contributes to the procoagulant role of Zn2+ in platelet-dependent fibrin formation.

Role of Platelet Glycoprotein VI and Tyrosine Kinase Syk in Thrombus Formation on Collagen-Like Surfaces.

Platelet interaction with collagens, via von Willebrand factor, is a potent trigger of shear-dependent thrombus formation mediated by subsequent engagement of the signaling collagen receptor glycoprotein (GP)VI, enforced by integrin α2ß1. Protein tyrosine kinase Syk is central in the GPVI-induced signaling pathway, leading to elevated cytosolic Ca2+. We aimed to determine the Syk-mediated thrombogenic activity of several collagen peptides and (fibrillar) type I and III collagens. High-shear perfusion of blood over microspots of these substances resulted in thrombus formation, which was assessed by eight parameters and was indicative of platelet adhesion, activation, aggregation, and contraction, which were affected by the Syk inhibitor PRT-060318. In platelet suspensions, only collagen peptides containing the consensus GPVI-activating sequence (GPO)n and Horm-type collagen evoked Syk-dependent Ca2+ rises. In whole blood under flow, Syk inhibition suppressed platelet activation and aggregation parameters for the collagen peptides with or without a (GPO)n sequence and for all of the collagens. Prediction models based on a regression analysis indicated a mixed role of GPVI in thrombus formation on fibrillar collagens, which was abolished by Syk inhibition. Together, these findings indicate that GPVI-dependent signaling through Syk supports platelet activation in thrombus formation on collagen-like structures regardless of the presence of a (GPO)n sequence.