Characterization of the Procoagulant Activity of Two Different Microparticle Species.

Abstract Abstract 1143 Background: Microparticles (MP) exhibit procoagulant activity by exposure of tissue factor (TF) and consecutive factor (F) × activation via binding to activated FVII (FVIIa). We have shown that TF-negative MP derived from endothelial cells (EMP) after exposure to cisplatin induce thrombin generation in vitro in a TF-independent manner (Lechner et al., J. Thromb. Haemost. 2007). The procoagulant properties of TF-negative MP are not well characterized. We therefore aimed to investigate the mechanisms of coagulation activation by TF-negative EMP (obtained from human pulmonary microvascular endothelial cells - HMVEC-L) in comparison to TF-abundant MP derived from a cancer cell line (A431-MP). Methods: MP were obtained by ultracentrifugation (100,000 × g for 1 h) of cell culture supernatant. The procoagulant activity was measured in normal plasma and in plasmas deficient in coagulation factors VII, VIII, IX, X, XI, and XII, respectively, by an in vitro thrombin generation assay (Technothrombin TGA, Technoclone, Austria), by activated partial thromboplastin (aPTT), prothrombin time (PT) and a chromogenic FIX activation assay. Antibodies to TF and FVII (both American Diagnostica, USA) were used. Plasmas used in the thrombin generation assay were MP-depleted by ultracentrifugation before use. Results: EMP and A431-MP induced in vitro thrombin generation in normal plasma. In vitro thrombin generation induced by EMP was abolished in plasmas deficient in FVIII or FIX, and was markedly reduced in FXI-deficient plasma. In FVII-deficient plasma, normal thrombin generation after addition of EMP was seen. In contrast, A431-MP-triggered thrombin generation was abolished in FVII-deficient plasma, but was not influenced by depletion of FVIII, FIX or FXI. In FX-deficient plasma, thrombin generation could not be triggered by addition of either EMP or A431-MP. Thrombin generation was normal after addition of EMP or A431-MP to FXII-deficient plasma (Figure). In a modified aPTT system using kaolin as surface activator, EMP and A431-MP induced clot formation in normal plasma. In a modified PT system only A431-MP but not EMP induced clot formation. We then investigated whether MP can directly activate FIX. In a plasma-free environment FIX activation by A431-MP was much more pronounced than by EMP (14-fold vs. 2.4 fold increase compared to a control experiment with cell culture medium only). FIX activation by A431-MP was blocked by the addition of antibodies to TF and FVII, while no such effect was seen after addition to EMP. Conclusions: Our findings show that both TF-positive and TF-negative MP exhibit procoagulant activity. If TF is expressed (A431-MP), coagulation activation is triggered via FVII/FVIIa, which is abolished in FVII-deficient plasma or by the addition of antibodies to TF or FVII. TF-negative EMP induce thrombin generation via the intrinsic coagulation pathway by activating FXI and FIX, as shown by the generation of activated FIX in a FIX activation assay. EMP are also able to induce clot formation in an aPTT based system. We surmise that the procoagulatory effect of EMP may be due to their high phospholipid content or to a specific phospholipid surface composition. The results of our FIX activation assay also confirm a direct FIX activation by TF/FVIIa, which can be abolished by addition of antibodies to TF or FVII. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Impact of V617F JAK2 Mutation on Monocyte Tissue Factor and Procoagulant Activity in Patients with Essential Thrombocythemia(ET) or Polycythemia VERA (PV)

Blood ◽

10.1182/blood.v112.11.3736.3736 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3736-3736

Author(s):

Anna Falanga ◽

Alfonso Vignoli ◽

Marina Marchetti ◽

Laura Russo ◽

Marina Panova-Noeva ◽

...

Keyword(s):

Tissue Factor ◽

Blood Coagulation ◽

Thrombin Generation ◽

Procoagulant Activity ◽

Jak2v617f Mutation ◽

Wild Type ◽

Coagulation Activation ◽

Allele Burden ◽

Generation Capacity

Abstract Clinical data suggest an increased thrombotic risk in patients with ET or PV carrying the JAK2V617F mutation. Laboratory data from our group show that ET patients carrying the JAK2V617F mutation are characterized by an enhanced platelet and neutrophil activation status (Falanga et al, Exp Hem 2007) and blood coagulation activation (Marchetti et al, Blood 2008), as compared to JAK2 wild-type ET. Since monocytes significantly contribute to blood coagulation activation as an important source of circulating tissue factor (TF), in this study we aimed to characterize the prothrombotic phenotype of monocytes from ET and PV patients and to evaluate whether and to what extent it is influenced by the JAK2V617F mutation. Twenty-four ET patients (10 JAK2 wild-type; 14 JAK2V617F carriers with 2%–35% mutant allele burden), 27 PV patients (all JAK2V617F carriers, 16 with 9%– 44% and 11 with 60%–100% allele burden, respectively), and 20 age-matched healthy subjects (controls, C) were enrolled into the study. Monocyte-associated TF antigen was measured on the cell surface by whole blood flow-cytometry, in both basal condition and after in vitro stimulation by bacterial endotoxin (lypopolysaccharide, LPS), as well as in cell lysates by ELISA. Monocyte procoagulant activity was evaluated by the Calibrated Automated Thrombogram (CAT) as the capacity of isolated monocyte lysates to induce thrombin generation in normal pool plasma. In basal conditions, significantly (p<0.05) higher surface levels of TF were measured on monocytes from ET (17.1±3.2% positive cells) and PV (24.4±3.7% positive cells) patients compared to C (8.2±1.9% positive cells). Similarly, the total TF antigen content of cell lysates was significantly increased in patients compared to C. The analysis of the data according to JAK2V617F mutational status, showed a gradient of increased TF expression starting from JAK2V617F negative patients (11.7±2.5%), versus JAK2V617F ET and PV subjects with <50% allele burden (20.3±3.6% and 23.2±2.8%, respectively), versus JAK2V617F PV patients with >50% allele burden (26.1±4.2%). The in vitro LPS stimulation significantly increased TF expression on monocytes from all study subjects and C compared to non-stimulated monocytes (p<0.05 for all groups), with a more elevated expression by monocytes from PV and ET patients compared to C. However, the relative increase in TF expression was greater in C (=3.7 fold) compared to both ET (=2.2 fold) and PV (=2 fold) patients. As observed in basal conditions, LPS-induced TF was higher in JAK2V617F positive patients as compared to negative, with the highest expression in JAK2V617F PV carriers with >50% allele load. Thrombin generation induced by monocytes from ET and PV patients was significantly increased compared to controls, as determined by significantly higher thrombin peaks (ET=145±12, PV=142±17, C=72.2±5 nM), and quantity of thrombin generated in time, i.e. the endogenous thrombin potential (ETP) (ET=1143±34, PV=1074±64, C=787±58 nM*min). The JAK2V617F PV subjects with >50% allele burden presented with the highest thrombin generation capacity (peak= 184±34 nM; ETP= 1268±32 nM). Our data indicate that the expression of the JAK2V617F mutation in ET and PV patients may confer to monocytes a different hemostatic phenotype in terms of increased expression of surface TF and thrombin generation capacity. These findings are in agreement with the previous observation of a hypercoagulable state associated with this mutation and suggest a new mechanism linking hemostatic cellular phenotypic alteration to genetic dysfunction in patients with myeloproliferative disease.

Download Full-text

Activation of Factor XI and the Contact Proteases by Products of Prothrombin Activation.

Blood ◽

10.1182/blood.v116.21.1150.1150 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1150-1150

Author(s):

Anton Matafonov ◽

Suryakala Sarilla ◽

Mao-fu Sun ◽

John P. Sheehan ◽

Vladimir Serebrov ◽

...

Keyword(s):

Amino Acids ◽

Thrombin Generation ◽

Conflicts Of Interest ◽

Fibrin Clot ◽

Clot Formation ◽

Clotting Time ◽

Contact Phase ◽

Thrombin Generation Assay ◽

Fibrinogen Binding ◽

Activation Assay

Abstract Abstract 1150 During plasma coagulation the protease α-thrombin (αIIa) cleaves fibrinogen to form a fibrin clot. Conversion of prothrombin to αIIa is catalyzed by factor (f) Xa, and results in expression of two electropositive regions on αIIa designated anion binding exosites (ABE) I and II. ABE I is involved in fibrinogen binding. In the presence of fVa and phospholipid, fXa cleaves prothrombin preferentially after Arg320, generating the intermediate meizothrombin (MzIIa), which also expresses ABE I. MzIIa is rapidly converted to αIIa. αIIa can be converted to β-thrombin (βIIa) and γ-thrombin (γIIa), both of which are cleaved within ABE I, and have greatly reduced capacity to convert fibrinogen to fibrin. Physiologic functions for βIIa or γIIa are not established; however, both have been identified in clotting blood. αIIa up-regulates its own generation in plasma by converting fXI to the protease fXIa. Yun et al. (J Biol Chem 2003;278:48112) showed that amino acids in ABE I are required for optimal fXI activation in the presence of the polyanion dextran sulfate (DS). MzIIa also activates fXI, consistent with a role for ABE I in protease binding to fXI. Given the absence of ABE I in βIIa and γIIa, it seems reasonable to postulate these proteases would interact poorly with fXI. In a clotting assay in which thrombin is added to plasma anticoagulated with citrate (low calcium), βIIa (12.5 nM) and γIIa (50 nM) did not induce clot formation, consistent with their low capacity to cleave fibrinogen. However, when plasma was recalcified to allow thrombin to form from endogenous prothrombin, both βIIa and γIIa induced clot formation. Recalcified plasma in the absence of βIIa or γIIa did not clot (800 sec observation period), indicating fibrin formation was βIIa/γIIa-dependent. Addition of an antibody to fXI prolonged the clotting time with βIIa, and prevented clotting with γIIa, suggesting βIIa and γIIa were activating fXI. In addition, with γIIa, a fXIIa inhibitor modestly prolonged clotting time, indicating the plasma contact phase was activated. We studied fXI activation by thrombin using western blot. βIIa and γIIa activated fXI at approximately half the rate of αIIa, while MzIIa activated fXI ∼4 fold faster than αIIa. FXI activation by αIIa is greatly enhanced by DS. In the presence of DS, αIIa and βIIa activated fXI comparably, while results with γIIa were not informative because the protease does not interact well with DS. Importantly, fXI activation by αIIa was not affected by the ABE I blocking peptide hirugen, indicating ABE I is not required for fXI activation by thrombin. While βIIa and γIIa were less effective fXI activators than αIIa and MzIIa in solution, significantly different results were obtained in a plasma thrombin generation assay. Here coagulation is initiated in fXII deficient plasma with thrombin (10 nM), and subsequent thrombin generation from endogenous prothrombin is monitored. The system is fXI-dependent, as a fXI antibody blocks thrombin generation. Prior work with this system indicates fXI is probably converted to fXIa by the thrombin added to initiate the process. Initiation of coagulation with αIIa and MzIIa resulted in comparable thrombin generation (∼250 nM). βIIa and γIIa, as well as recombinant αIIa with mutations in amino acids in ABE I induced thrombin generation ∼2-fold greater than for αIIa and MzIIa. We hypothesized this was due to the inability of fibrinogen to compete with fXI for binding to thrombin species lacking ABE I. Consistent with this, hirugen peptide enhanced αIIa initiated thrombin generation ∼4-fold. Finally, we followed up on the observation that a fXIIa inhibitor prolonged time to γIIa-induced clot formation in recalcified plasma. In solution, γIIa, but not αIIa, βIIa, or MzIIa cleaves the contact factors fXII and PK. The cleaved proteases, in turn, are capable of cleaving chromogenic substrates, and have activity in a reciprocal fXII-PK activation assay. Our studies show that ABE I is not required for thrombin-mediated activation of fXI, that thrombin species not fully expressing ABE I may be better than αIIa and MzIIa as initiators of fXI-dependent thrombin generation in plasma, and that γIIa can activate the plasma contact proteases. Taken as a whole, the data indicate forms of thrombin other than αIIa may contribute directly to feedback activation of fXI, and may represent a previously unrecognized link between coagulation and the contact system. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Evaluation of the Antithrombin Potential in a Thrombin Generation Assay Performed at the Surface of Endothelial Cells

Blood ◽

10.1182/blood.v124.21.4168.4168 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4168-4168

Author(s):

Béatrice Catieau ◽

Sami Chtourou ◽

Jean-Luc Plantier

Keyword(s):

Endothelial Cells ◽

Thrombin Generation ◽

Peak Height ◽

Factor Ix ◽

Flat Bottom ◽

Thrombin Generation Assay ◽

Heparan Sulfates ◽

Vitro Effect ◽

Control Plasma

Abstract Thrombin generation assay (TGA) was recently evaluated on a living endothelial-derived cell line (Coll et al. J. Thromb. Haemost. 2013; 11, 1916). This innovative assay brought into an hemostasis assay the cellular components of the anticoagulation pathway (APC and TFPI pathways) as well as a activated cell surface. It might help elucidate the relationship between hemostasis and inflammation in a more complex system. In the aim of evaluating the potential of antithrombin (AT) connecting both processes we set-up a similar assay on human vein endothelial cells (HUVEC). We first demonstrated that thrombin generation can be measured in flat-bottom 96-wells in factor IX-or factor VIII-deficient plasma substituted by either 0.1 or 1 U/ml of FIX or FVIII, respectively. Next, HUVEC were grown and expanded in a complete commercial medium (EndoGRO-LS, Millipore) for no more than 6 passages. Wells were then coated with gelatin 1% and cells seeded at 10,000 cells/well. The binding of plasma-derived AT (Aclotine ®, LFB; France dialyzed in cell culture medium) to HUVEC was demonstrated as being dose- (0.5; 1; 2.5 and 5 U/ml) and time- (0-6 hours) dependent. Saturating conditions were found using 2.5 U/ml AT for a 2h incubation. We also showed that the binding was moderately affected in the presence of heparin at concentrations up to 50 U/ml (loss of 19% of the signal) and not at all following an heparanase I+II+III treatment suggesting that another receptor(s) than cellular heparan sulfates being responsible for this interaction. The effect of AT on coagulation was then compared in the presence of cells or not. To do this cells were grown to confluence, washed with non-supplemented medium and incubated in the presence of the TGA mix (plasma containing AT or not, 0.5 pM Tissue Factor, 4 µM Phospholipids). The reaction was initiated by injection of the FluCa kit thrombin substrate (Stago). In the presence of HUVEC, the efficiency of thrombin generation from a control plasma (Unicalibrator, Stago) was decreased with a lag time increased (from 5.67 min to 6.83 min), the peak height diminished from 204.4 nM thrombin to 150.4 nM and the velocity from 55.8 nM/min to 33.4 nM/min. However, the overall amount of thrombin generated was less affected, diminishing from 1515.5 nM to 1482 nM. These data confirms that the presence of the HUVEC anticoagulants pathways can effectively diminish the thrombin generation. Without cells, the presence of 0.5, 1 or 2 U/ml AT dose-dependently decreased the generation of thrombin from the control plasma. The velocity was decreased by 23.2%, 57.6% and 75.5% and the peak height by 33.5%, 61.5% and 78.8%, respectively. When the same experiment was performed in the presence of HUVEC cells, the concentrations of AT similarly decreased the velocity by 34.2%, 54% and 70 % and the peak height by 39%, 59.1% 74.3%, respectively. There was no difference in the TGA parameters if AT was pre-incubated at the surface of the cells for up to 2h prior the TGA or if it was added extemporaneously. These results indicate that the presence of HUVEC did not modulate the in vitro effect of AT during coagulation. The effect of AT on the cell response during this process are in the process of being investigated with a particular focus on the anti-inflammatory properties of AT. Disclosures Catieau: LFB Biotechnologies: Employment. Chtourou:LFB Biotechnologies: Employment. Plantier:LFB Biotechnologies: Employment.

Download Full-text

Cellular origin and procoagulant properties of microparticles in meningococcal sepsis

Blood ◽

10.1182/blood.v95.3.930.003k46_930_935 ◽

2000 ◽

Vol 95 (3) ◽

pp. 930-935 ◽

Cited By ~ 376

Author(s):

Rienk Nieuwland ◽

René J. Berckmans ◽

Sarah McGregor ◽

Anita N. Böing ◽

Fred P. H. Th. M. Romijn ◽

...

Keyword(s):

Thrombin Generation ◽

Enzyme Linked Immunosorbent Assay ◽

Meningococcal Sepsis ◽

Coagulation Activation ◽

Cellular Origin ◽

Thrombin Generation Assay ◽

Circulating Microparticles ◽

Clinical Conditions

Patients with meningococcal sepsis generally suffer from disseminated intravascular coagulation (DIC). The aim of this study was to address whether these patients have elevated numbers of circulating microparticles that contribute to the development of DIC. Plasma samples from 5 survivors, 2 nonsurvivors, and 5 healthy volunteers were analyzed for the presence of microparticles by flow cytometry. Ongoing coagulation activation in vivo was quantified by enzyme-linked immunosorbent assay of plasma prothrombin fragment F1 + 2, and procoagulant properties of microparticles in vitro were estimated by thrombin-generation assay. On admission, all patients had increased numbers of microparticles originating from platelets or granulocytes when compared with controls (P = .004 and P = .008, respectively). Patients had elevated levels of F1 + 2 (P = .004), and their microparticles supported thrombin generation more strongly in vitro (P = .003) than those of controls. Plasma from the patient with the most fulminant disease course and severe DIC contained microparticles that expressed both CD14 and tissue factor, and these microparticles demonstrated extreme thrombin generation in vitro. We conclude that patients with meningococcal sepsis have elevated numbers of circulating microparticles that are procoagulant. These findings may suggest a novel therapeutic approach to combat clinical conditions with excessive coagulation activation.

Download Full-text

Inactivated antithrombins as fondaparinux antidotes: a promising alternative to haemostatic agents as assessed in vitro in a thrombin-generation assay

Thrombosis and Haemostasis ◽

10.1160/th15-12-0927 ◽

2016 ◽

Vol 116 (09) ◽

pp. 452-460 ◽

Cited By ~ 2

Author(s):

Yasmine Bourti ◽

Judicael Fazavana ◽

Marine Armand ◽

François Saller ◽

Dominique Lasne ◽

...

Keyword(s):

Thrombin Generation ◽

Anticoagulant Activity ◽

Factor Vii ◽

Recombinant Activated Factor Vii ◽

Promising Alternative ◽

Thrombin Generation Assay ◽

Normal Plasma ◽

Haemostatic Agents ◽

Heparin Derivatives

SummaryIn the absence of specific antidote to fondaparinux, two modified forms of antithrombin (AT), one recombinant inactive (ri-AT) and the other chemically inactivated (chi-AT), were designed to antagonise AT-mediated anticoagulants, e. g. heparins or fondaparinux. These inactive ATs were previously proven to effectively neutralise anticoagulant activity associated with heparin derivatives in vitro and in vivo, as assessed by direct measurement of anti-FXa activity. This study was undertaken to evaluate in vitro the effectivity of inactive ATs to reverse anticoagulation by heparin derivatives and to compare them with non-specific fondaparinux reversal agents, like recombinant-activated factor VII (rFVIIa) or activated prothrombin-complex concentrate (aPCC), in a thrombin-generation assay (TGA). Addition of fondaparinux (3 μg/ml) to normal plasma inhibited thrombin generation by prolonging lag time (LT) as much as 244 % and lowering endogenous thrombin potential (ETP) to 17 % of their control (normal plasma) values. Fondaparinux-anticoagulant activity was reversed by ri-AT and chi-AT, as reflected by the corrections of LT up to 117 % and 114 % of its control value, and ETP recovery to 78 % and 63 %, respectively. Unlike ri-AT that had no effect on thrombin generation in normal plasma, chi-AT retained anticoagulant activity that minimises its reversal capacity. However, both ATs were more effective than rFVIIa or aPCC at neutralising fondaparinux and, unlike non-specific antidotes, inactive ATs specifically reversed AT-mediated anticoagulant activities, as suggested by their absence of procoagulant activity in anticoagulant-free plasma.

Download Full-text

Desmopressin (DDAVP) Enhances Platelet Adhesion to the Extracellular Matrix of Cultured Human Endothelial Cells through Increased Expression of Tissue Factor

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656088 ◽

1997 ◽

Vol 77 (05) ◽

pp. 0975-0980 ◽

Cited By ~ 23

Author(s):

Angel Gálvez ◽

Goretti Gómez-Ortiz ◽

Maribel Díaz-Ricart ◽

Ginés Escolar ◽

Rogelio González-Sarmiento ◽

...

Keyword(s):

Extracellular Matrix ◽

Endothelial Cells ◽

Tissue Factor ◽

Platelet Adhesion ◽

Umbilical Vein ◽

Procoagulant Activity ◽

Experimental Conditions ◽

Chromogenic Assay

SummaryThe effect of desmopressin (DDAVP) on thrombogenicity, expression of tissue factor and procoagulant activity (PCA) of extracellular matrix (ECM) generated by human umbilical vein endothelial cells cultures (HUVEC), was studied under different experimental conditions. HUVEC were incubated with DDAVP (1, 5 and 30 ng/ml) and then detached from their ECM. The reactivity towards platelets of this ECM was tested in a perfusion system. Coverslips covered with DD A VP-treated ECMs were inserted in a parallel-plate chamber and exposed to normal blood anticoagulated with low molecular weight heparin (Fragmin®, 20 U/ml). Perfusions were run for 5 min at a shear rate of 800 s1. Deposition of platelets on ECMs was significantly increased with respect to control ECMs when DDAVP was used at 5 and 30 ng/ml (p <0.05 and p <0.01 respectively). The increase in platelet deposition was prevented by incubation of ECMs with an antibody against human tissue factor prior to perfusion. Immunofluorescence studies positively detected tissue factor antigen on DDAVP derived ECMs. A chromogenic assay performed under standardized conditions revealed a statistically significant increase in the procoagulant activity of the ECMs produced by ECs incubated with 30 ng/ml DDAVP (p <0.01 vs. control samples). Northern blot analysis revealed increased levels of tissue factor mRNA in extracts from ECs exposed to DDAVP. Our data indicate that DDAVP in vitro enhances platelet adhesion to the ECMs through increased expression of tissue factor. A similar increase in the expression of tissue factor might contribute to the in vivo hemostatic effect of DDAVP.

Download Full-text

The role of matrix metalloproteinase activity in the maturation of human capillary endothelial cells in vitro

Journal of Cell Science ◽

10.1242/jcs.112.10.1599 ◽

1999 ◽

Vol 112 (10) ◽

pp. 1599-1609 ◽

Cited By ~ 2

Author(s):

B.M. Kraling ◽

D.G. Wiederschain ◽

T. Boehm ◽

M. Rehn ◽

J.B. Mulliken ◽

...

Keyword(s):

Cell Culture ◽

Cell Proliferation ◽

Endothelial Cells ◽

Endothelial Cell ◽

Endothelial Cell Proliferation ◽

Protein Levels ◽

Matrix Deposition ◽

Capillary Endothelial Cells ◽

Vessel Maturation

Vessel maturation during angiogenesis (the formation of new blood vessels) is characterized by the deposition of new basement membrane and the downregulation of endothelial cell proliferation in the new vessels. Matrix remodeling plays a crucial, but still poorly understood role, in angiogenesis regulation. We present here a novel assay system with which to study the maturation of human capillary endothelial cells in vitro. When human dermal microvascular endothelial cells (HDMEC) were cultured in the presence of dibutyryl cAMP (Bt2) and hydrocortisone (HC), the deposition of a fibrous lattice of matrix molecules consisting of collagens type IV, type XVIII, laminin and thrombospondin was induced. In basal medium (without Bt2 and HC), HDMEC released active matrix metalloproteinases (MMPs) into the culture medium. However, MMP protein levels were significantly reduced by treatment with Bt2 and HC, while protein levels and activity of endogenous tissue inhibitor of MMPs (TIMP) increased. This shift in the proteolytic balance and matrix deposition was inhibited by the specific protein kinase A inhibitors RpcAMP and KT5720 or by substituting analogues without reported glucocorticoid activity for HC. The addition of MMP inhibitors human recombinant TIMP-1 or 1,10-phenanthroline to cultures under basal conditions induced matrix deposition in a dose-dependent manner, which was not observed with the serine protease inhibitor epsilon-amino-n-caproic acid (ACA). The deposited basement membrane-type of matrix reproducibly suppressed HDMEC proliferation and increased HDMEC adhesion to the substratum. These processes of matrix deposition and downregulation of endothelial cell proliferation, hallmarks of differentiating new capillaries in the end of angiogenesis, were recapitulated in our cell culture system by decreasing the matrix-degrading activity. These data suggest that our cell culture assay provides a simple and feasible model system for the study of capillary endothelial cell differentiation and vessel maturation in vitro.

Download Full-text

Vascular repair utilising immobilised heparin conjugate for protection against early activation of inflammation and coagulation

Thrombosis and Haemostasis ◽

10.1160/th14-09-0724 ◽

2015 ◽

Vol 113 (06) ◽

pp. 1312-1322 ◽

Cited By ~ 18

Author(s):

Sofia Nordling ◽

Jaan Hong ◽

Karin Fromell ◽

Fredrik Edin ◽

Johan Brännström ◽

...

Keyword(s):

Endothelial Cells ◽

Thrombus Formation ◽

Innate Immune ◽

Human Endothelial Cells ◽

Coagulation Activation ◽

Ischaemia Reperfusion Injury ◽

Novel Approach ◽

One Step ◽

Chamber Model

SummaryIschaemia-reperfusion injury (IRI) poses a major challenge in many thrombotic conditions and in whole organ transplantation. Activation of the endothelial cells and shedding of the protective vascular glycocalyx during IRI increase the risk of innate immune activation, cell infiltration and severe thrombus formation, promoting damage to the tissue. Here, we present a novel one-step strategy to protect the vasculature by immobilisation of a unique multi-arm heparin conjugate to the endothelium. Applying a new in vitro blood endothelial cell chamber model, the heparin conjugate was found to bind not only to primary human endothelial cells but also directly to the collagen to which the cells adhered. Incubation of hypoxic endothelial cells with freshly drawn human blood in the blood chambers elicited coagulation activation reflected by thrombin anti-thrombin formation and binding of platelets and neutrophils. Immobilisation of the heparin conjugate to the hypoxic endothelial cells created a protective coating, leading to a significant reduction of the recruitment of blood cells and coagulation activation compared to untreated hypoxic endothelial cells. This novel approach of immobilising multi-arm heparin conjugates on the endothelial cells and collagen of the basement membrane ensures to protect the endothelium against IRI in thrombotic disorders and in transplantation.

Download Full-text