Coagulation on Endothelial Cells: The Underexposed Part of Virchow’s Triad

2012 ◽  
Vol 108 (11) ◽  
pp. 863-871 ◽  
Author(s):  
Irma Geenen ◽  
Mark Post ◽  
Daniel Molin ◽  
Geert Schurink ◽  
Jos Maessen ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Thrombin Generation ◽  
Factor Viia ◽  
Bypass Graft ◽  
Peak Height ◽  
Factor Xii ◽  
Contact Activation ◽  
Extrinsic Pathway ◽  
Serum Free ◽  
Cabg Patients

SummaryThe process of thrombin generation involves numerous plasma proteases and cofactors. Interaction with the vessel wall, in particular endothelial cells (ECs), influences this process but data on this interaction is limited. We evaluated thrombin generation on EA.hy926, human coronary arterial ECs (HCAECs) and patient-derived human venous ECs (HVECs) by means of a modified calibrated automated thrombogram (CAT) method and especially looked into contribution of the intrinsic and extrinsic pathways. Thrombin generation was measured in presence of confluent ECs with normal pooled and factor XII-deficient (FXII-deficient) platelet-poor plasma, with/without active site inhibited factor VIIa (ASIS) to block the extrinsic pathway and corn trypsin inhibitor for blocking contact activation (intrinsic pathway). Fetal bovine serum (FBS) was removed from culture conditions as FXIIa from the serum retained on ECs apparently, thereby inducing strong contact activation. In serum-free conditions, EA.hy926 and patient-derived HVECs induced thrombin generation mainly via the contact activation pathway with minor influence of ASIS on peak height and very low thrombin generation curves in FXII-deficient plasma. HVECs derived from coronary arterial bypass graft (CABG) patients showed increased thrombin generation compared to control patients, which could be ascribed to increased contact activation. Contribution of the extrinsic pathway on patient-derived ECs was limited. We conclude that the CAT method in combination with serum-free cultured ECs offers a valuable high-throughput method to evaluate endothelial influences on thrombin generation, which appears to involve predominantly contact activation on ECs. Contact activation-mediated thrombin generation was increased on ECs from CABG patients compared to controls.

Monitoring Recombinant FVIIa in Hemophilic and Normal Plasma Using a Thrombin Generation Assay

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1207-1207
Author(s):  
Sarah T.B.G. Loubele ◽  
Henri M.H. Spronk ◽  
Rene van Oerle ◽  
Hugo ten Cate ◽  
Peter L.A. Giesen
Keyword(s):  
Thrombin Generation ◽  
Factor Viia ◽  
Optimal Dose ◽  
Peak Height ◽  
Contact Activation ◽  
Mean Values ◽  
Thrombin Generation Assay ◽  
Normal Plasma ◽  
The Mean ◽  
20 Nm

Abstract Abstract 1207 Background: Although the recombinant factor VIIa (rFVIIa) has been registered for use in hemophilia patients with inhibitors, there is still no method to monitor the effects of rFVIIa in restoring the coagulation balance in plasma. Hence, information is lacking about the individual optimal dose needed to normalize thrombin generation. Methods: The calibrated automated thrombogram (CAT) method was modified to increase sensitivity for rFVIIa addition to plasma at concentrations of 0, 2.5, 5, 10, 20, 40 and 80 nM, which covers the expected plasma concentration of 26 nM reached after standard administration. Thrombin generation was triggered using combinations of TF concentrations between 0 and 4 pM, and phospholipids concentrations between 0 and 4 μM. Endogenous thrombin potential (ETP), peak height, and velocity index were calculated in platelet poor plasmas (PPP) of different donors. All blood was collected in citrated tubes containing corn trypsin inhibitor (CTI) to minimize any contact activation. Results: The optimal conditions for discriminating rFVIIa (0–80 nM) in the CAT assay were determined in PPP: 0 or 0.25 pM TF with 4 μM of phospholipids. Also at higher TF concentrations, the CAT method was able to detect varying rFVIIa concentrations. The optimal concentration of phospholipids was 4 μM for all TF concentrations. In plasma of 6 healthy volunteers, thrombin generation triggered with 0.25 pM TF dose dependently increased using varying rFVIIa concentrations between 0 and 80 nM (Figure 1, left panel). The mean values for ETP, peak height and velocity index are depicted in Table 1. On average, addition of 2.5, 5, 10, 20, 40 or 80 nM of rFVIIa resulted in a 146, 156, 161, 174, 206, and 285 % of the peak height compared to 0 nM rFVIIa, which was set at 100 %. At 4 pM TF the maximum ETP, peak height, and velocity index were reached at concentrations less than 20 nM rFVIIa for all donors. The mean values are depicted in Table 2. Surprisingly, at 80 nM rFVIIa, thrombin generation was decreased compared to lower rFVIIa concentrations (Figure 1, right panel). Addition of 2.5, 5, 10, 20, 40 or 80 nM of rFVIIa resulted in 107, 109, 107, 103, 100, or 94 % of the peak height without addition of rFVIIa (0 nM set at 100 %). In FVIII deficient patient plasma (PPP), this effect was also present and even more pronounced. Here, a dose dependent effect of rFVIIa addition was visible at low (0 or 0.25 pM) TF trigger, whereas at 4 pM TF trigger ETP, peak height and velocity index were maximal in the presence of 10 nM rFVIIa. Overall, the peak height was 136, 142, 126, 102, 94, and 81 % upon addition of 5, 10, 20, 30, 40, or 80 nM rFVIIa respectively compared to 0 nM rFVIIa (set to 100 %). Discussion: In hemophilic as well as normal plasma, the addition of rFVIIa dose dependently altered thrombin generation triggered with a low TF trigger (0 or 0.25 pM). At a higher trigger of 4 pM TF, maximal thrombin generation was obtained at rFVIIa concentrations of less then 20 nM. Remarkably, thrombin generation was attenuated in the presence of 80 nM rFVIIa. This paradox may be explained by assuming that the endogenously activated VIIa is more active than the rVIIa that was added. At higher rVIIa dosages the fraction of TF occupied by endogenous VIIa will decrease resulting in less active TF:VIIa complexes. This effect will be more pronounced when FXa formation is dependent on TF:FVIIa alone without the involvement of the tenase complex, which shows from our analysis in hemophilic plasma. Overall, these data suggest that the assay is most sensitive to added rVIIa when the contribution to Xa formation of TF:VIIa complex is small compared to that of rVIIa alone, i.e., in conditions where there is no or very little TF present. Disclosures: Giesen: Thrombinoscope bv: Employment.

Activity and Regulation of Factor VIIa Analogs with Increased Potency at the Endothelial Cell Surface.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1751-1751
Author(s):  
Samit Ghosh ◽  
Mirella Ezban ◽  
Egon Persson ◽  
Ulla Hedner ◽  
Usha Pendurthi ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Surface ◽  
Proteolytic Activity ◽  
Thrombin Generation ◽  
Factor Viia ◽  
Factor Xa ◽  
Factor Ix ◽  
Factor X ◽  
Wild Type

Abstract High doses of recombinant factor VIIa (FVIIa) have been found to bypass factor IX or factor VIII deficiency and ameliorate the bleeding problems associated with hemophilia patients with inhibitors. Recent studies show that FVIIa also acts as an effective hemostatic agent in other categories of patients, and thus has become a promising candidate for prevention and treatment of excessive bleeding associated with many other diseases/injuries. Although recombinant FVIIa has proven to be a very effective and safe drug in the treatment of bleeding episodes in hemophilia patients with inhibitors and other indications, a small fraction of patients may be refractory to FVIIa treatment. The reason for this is unclear at present, but it is possible that administration of very high pharmacological doses of FVIIa or use of genetically modified FVIIa molecules with increased potencies may circumvent the problem. The most dramatic effect on the activity (a 40-fold increase in proteolytic activity) of FVIIa was obtained by occupying the corresponding positions in thrombin/factor IXa for those positions 158, 296 and 298 of FVIIa (FVIIaDVQ). A FVIIa mutant in which the hydrophobic residue Met 298 was replaced with Gln (FVIIaQ) has 7-fold higher proteolytic activity. In the present study, we investigated the interactions of FVIIaQ and FVIIaDVQ with plasma inhibitors, tissue factor pathway inhibitor (TFPI) and antithrombin (AT) in solution and at the vascular endothelium. Both TFPI and AT/heparin inhibited the FVIIa variants more rapidly than the wild-type FVIIa in the absence of TF. In the presence of TF, TFPI, TFPI-Xa and AT/heparin inhibited FVIIa and FVIIa variants at similar rates. Although the wild-type FVIIa failed to generate significant amounts of factor Xa on unperturbed endothelial cells, FVIIa variants, particularly FVIIaDVQ, generated a substantial amount of factor Xa on unperturbed endothelium (1 nM of factor VIIa generated 0.3 ± 0.15 nM factor Xa/h whereas FVIIaQ and FVIIaDVQ generated 1.26 ± 0.1 nM/h and 9.48 ± 1.32 nM/h, respectively). Annexin V fully attenuated the FVIIa-mediated activation of factor X on unperturbed endothelial cells whereas anti-TF IgG had no effect. On stimulated HUVEC, FVIIa and FVIIa variants activated factor X at similar rates (30–40 nM/h). AT/heparin and TFPI-Xa inhibited the activity of FVIIa and FVIIa variants bound to endothelial cell TF in a similar fashion. AT inhibition of FVIIa bound to stimulated endothelial cells requires exogenous heparin. Interestingly, TFPI-Xa was found to inhibit the activities of both FVIIa and FVIIa analogs bound to unperturbed endothelial cells. Despite significant differences observed in factor Xa generation on native endothelium exposed to FVIIa and FVIIa analogs, no differences were found in thrombin generation when cells were exposed to FVIIa or FVIIa analogs under plasma mimicking conditions, probably due to limited availability of anionic phospholipids and/or putative factor Xa and Va binding sites on their cell surface. Over all, our present data suggest that although FVIIa variants may generate factor Xa on native endothelium, the resultant factor Xa does not lead to enhanced thrombin generation on native endothelium compared to FVIIa. These data should reduce potential concerns about whether the use of FVIIa variants triggers unwanted coagulation on native endothelium, and may facilitate the development of FVIIa analogs as effective therapeutic agents in near future for treatment of patients with bleeding disorders.

Feedback Activation of Factor XI by Thrombin Is Essential for Hemostasis In Vivo.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2127-2127
Author(s):  
Henri M. H. Spronk ◽  
Sabine Wilhelm ◽  
Rene Van Oerle ◽  
Menno L. Knetsch ◽  
David Gailani ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Factor Viia ◽  
Fibrin Clot ◽  
Intrauterine Death ◽  
Factor Xii ◽  
Dependent Manner ◽  
Factor Xi ◽  
Feedback Activation

Abstract Abstract 2127 Poster Board II-102 Background: The revised model of coagulation proposes that factor XI (FXI) can be activated by thrombin, which is generated upon activation of the tissue factor (TF) pathway. This concept, however, has not been tested in vivo. A recent study questioned the existence of this feedback loop and suggested that factor XII (FXII) is the sole activator of FXI. Here, we analyze the feedback activation of FXI in plasma and in genetically altered mice. Methods and results: Fluorescence-based assays indicated that particle-bound thrombin caused thrombin generation in plasma both in the absence of TF and in the presence of active site inhibited factor VIIa. Thrombin failed to activate FXII and thrombin generation was almost completely abolished by an anti-FXIa antibody and in FXI-deficient plasma. Surface bound thrombin induced complex formation of FXI, with its major inhibitor C1 inhibitor, even in FXII-deficient plasma in a time and dose dependent manner. To determine if thrombin-driven FXI activation is important for hemostasis in vivo we used TF deficient mice (low TF), which have severely reduced thrombin formation. Low TF mice were crossed with mice deficient in one of the intrinsic pathway proteases FXII, FXI, or FIX. Double deficiency in TF and either FIX or FXI resulted in the intrauterine death of embryos due to hemorrhage. In contrast low TF/FXII-null mice were viable and the bleeding phenotype was unchanged from low TF animals. Conclusions: Surface-bound thrombin, a model for fibrin clot-protected thrombin, generates thrombin in a FXI dependent manner, independently from FXII. In addition to corroborating an amplifying role of FXI in thrombin generation, we provide the first evidence that at low TF levels FXI is essential in generating a sufficient ambient level of thrombin to permit embryonic development. Disclosures: No relevant conflicts of interest to declare.

Dual role of collagen in factor XII–dependent thrombus formation

Blood ◽  
2009 ◽  
Vol 114 (4) ◽  
pp. 881-890 ◽  
Author(s):  
Paola E. J. van der Meijden ◽  
Imke C. A. Munnix ◽  
Jocelyn M. Auger ◽  
José W. P. Govers-Riemslag ◽  
Judith M. E. M. Cosemans ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Type I Collagen ◽  
Factor Viia ◽  
Thrombus Formation ◽  
Dual Role ◽  
Type I ◽  
Factor Xii ◽  
Glycoprotein Vi

Abstract In vivo mouse models have indicated that the intrinsic coagulation pathway, initiated by factor XII, contributes to thrombus formation in response to major vascular damage. Here, we show that fibrillar type I collagen provoked a dose-dependent shortening of the clotting time of human plasma via activation of factor XII. This activation was mediated by factor XII binding to collagen. Factor XII activation also contributed to the stimulating effect of collagen on thrombin generation in plasma, and increased the effect of platelets via glycoprotein VI activation. Furthermore, in flow-dependent thrombus formation under coagulant conditions, collagen promoted the appearance of phosphatidylserine-exposing platelets and the formation of fibrin. Defective glycoprotein VI signaling (with platelets deficient in LAT or phospholipase Cγ2) delayed and suppressed phosphatidylserine exposure and thrombus formation. Markedly, these processes were also suppressed by absence of factor XII or XI, whereas blocking of tissue factor/factor VIIa was of little effect. Together, these results point to a dual role of collagen in thrombus formation: stimulation of glycoprotein VI signaling via LAT and PLCγ2 to form procoagulant platelets; and activation of factor XII to stimulate thrombin generation and potentiate the formation of platelet-fibrin thrombi.

scholarly journals The Relationship between Thrombin Generation Assay and FVIII Levels in Patients with Mild to Moderate Haemophilia (A)

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2454-2454
Author(s):  
Pu-Lin Luo ◽  
Steven K. Austin ◽  
Kiran Parmar ◽  
Dan P Hart ◽  
Michael Laffan
Keyword(s):  
Research Funding ◽  
Thrombin Generation ◽  
Haemophilia A ◽  
Contact Activation ◽  
Extrinsic Pathway ◽  
Two Stage ◽  
One Stage ◽  
Thrombin Generation Assay ◽  
The Relationship

Abstract Introduction Haemophilia A (HA) phenotypes (mild, moderate and severe) are based on the baseline FVIII levels, however considerable variation in the bleeding phenotype exists between patients with similar FVIII level. Moreover, approximately 40% of patients with mild HA have large discrepancies between FVIII measured by one stage (FVIII:C1) and two stage methods (FVIII:Chr2) and it is unclear which method correlates best with in vivo FVIII function and bleeding phenotype. The Thrombin Generation assay (TGA), a global measure of haemostasis may be a better predictor of bleeding phenotype but pre-analytical factors such as contact activation can confound the results. Choice of initiating conditions may also be critical in determining sensitivity: recent studies have suggested that initiation with FIXa rather than tissue factor (TF) in detecting low levels of FVIII:C in severe HA, however its utility in mild to moderate HA patients has yet to be determined. The aim of this study is to establish the relationship between FVIII:C and TGA and the influence of contact factor activation in TF and FIXa triggered TGA in patients with mild to moderate HA. Methods This is a prospective cohort study. Patients aged >18 with known congenital HA and FVIII:C 0.01- 0.2 iu/ml were recruited from 3 Haemophilia Comprehensive Care Centres in London. Peripheral blood was drawn into citrate Vacutainer tubes containing 0.106M trisodium citrate (1:9 volume) and Vacutainer tubes preloaded with CTI (50µg/ml). Samples underwent double centrifugation (2500g) to obtain platelet free plasma. Thrombin generation assay, using a standard calibrated automated thrombogram was triggered with either TF (1pmol) or FIXa (5nM). Factor FVIII levels were assessed by one stage APTT based (FVIII:C1) and two stage chromogenic (FVIII:Chr2) methods. Mutation analysis was carried out in all patients. Results 40 patients were recruited in the study. Five patients (13%) had standard FVIII discrepancy (FVIII:C1/FVIII:Chr2>1.5) with 4 different FVIII mutations located on the inter-domain surface of the A2 domain (p.Tyr683Ser, p.Arg550Cys, p.Gly498Arg, p.MET681.Le). One patient had reverse FVIII discrepancy. In TF triggered TGA, the presence of CTI resulted in significant reduction in mean ETP (nmol .min)(455. vs 278, p<0.01, 95% CI 104-243), mean Peak thrombin (nM) (37.81 vs 16.54, t(6.6) p<0.01 95%CI 14.7-27.3), and mean Velindex (nM/min) (4.86 vs 1.29 t(7.0), p<0.01, 95% CI2.3-4.19) and a longer mean ttPeak (min) (14.26 vs 16.22, t(-3.2) p=0.02 95% CI-3.1- -0.76). In contrast, the presence of CTI did not affect ETP (1143 vs 1042, p=0.19 95% CI -54-256), mean Peak thrombin (252 vs 251, p=0.6 95%CI 27-46) or Velindex (118.54 vs 119.15 p= 0.95, 95%CI -23-12.9) in FIXa triggered TGA. There was a good correlation between FVIII:Chr2 and ETP (r=0.56, p=<0.001) Peak (r=0.6, p=<0.001) and Velindex (r=0.7, p=<0.001) in TF(CTI-) triggered TGA, however no relationship was seen between FVIII:C and TG parameters (ETP r=-0.01 p=0.9, Peak r=-0.003, p=0.97 and Velindex r=0.018, p=0.9) in TF(CTI+) triggered TGA. In both FIXa(CTI-) and FIXa (CTI+) triggered TGA, there was a good correlation seen between Lagtime (r=-0.6 p=<0.01), Peak (r=0.4-0.6, p=<0.01) ttpeak (r= -0.6, p=<0.01) and Velindex (r=0.69 <0.01) with FVIII:Chr2 but not with ETP. In patients with standard FVIII discrepancy (n=5), their ETP and Peak levels in TF and FIXa triggered TGA were in keeping with the ETP and Peak levels of non-discrepant patients with similar FVIII:C2 and significantly lower than that of non-discrepant patients with similar FVIII:C1. Conclusions Our study confirms that at low TF triggered TG, contact factor activation in vitro is an important preanalytical variable. Curiously any TG correlation with FVIII level is lost once the contact pathway is inhibited suggesting that TG remains largely determined by the extrinsic pathway in this system. In contrast, factor FIXa triggered TG is unaffected by inhibition of contact activation and demonstrates a good correlation to FVIII:C with or without CTI. This can be explained by suggesting that the supply of FIXa negates any effect of XIa from contact activation and that TG by this route is more completely dependent on FVIII. Therefore a FIXa triggered TGA may offer a better alternative in the assessment of haemophilia and further studies are underway to determine whether this is a better predictor of bleeding phenotypes. Disclosures Luo: Pfizer: Research Funding. Austin:Pfizer: Research Funding. Laffan:Pfizer: Honoraria; Roche: Consultancy, Speakers Bureau.

Glycosylphosphatidylinositol Anchored TFPI As Main Regulator of Factor X Activation On the Surface of Endothelial Cells.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2210-2210
Author(s):  
Michael Dockal ◽  
Robert Pachlinger ◽  
Angelina Baldin-Stoyanova ◽  
Fabian Knofl ◽  
Nadja Ullrich ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Tissue Factor ◽  
Conflicts Of Interest ◽  
Factor Viia ◽  
Umbilical Vein ◽  
Surface Proteins ◽  
Enzyme Linked Immunosorbent Assay ◽  
Factor X ◽  
Extrinsic Pathway

Abstract Abstract 2210 Tissue factor pathway inhibitor (TFPI) is a key regulator of factor X (FX) activation in the extrinsic pathway of blood coagulation. TFPI inhibits FXa generation by formation of a quaternary complex consisting of factor VIIa (FVIIa), tissue factor (TF), FXa and TFPI. The main portion (∼80%) of TFPI in humans is reportedly associated with endothelial cells. We used human umbilical vein endothelial cells (HUVECs) as a model to obtain further insight into the function of TFPIα and the glycosylphosphatidylinositol (GPI) anchored TFPI form, which represents TFPIα bound to GPI-anchored surface proteins and/or TFPIβ. In contrast to TFPIα, which consists of 3 Kunitz domains (KD) and a basic C-terminal part, GPI-anchored TFPIβ lacks the third Kunitz domain (KD3) and the basic C–terminal region due to alternative splicing. In TFPIβ these two domains are replaced by a sequence that adds a GPI anchor to the protein linking it to the cell membrane. Treatment of HUVECs with phosphatidylinositol phospholipase C (PI-PLC) that cleaves GPI-anchors and subsequent fluorescence activated cell sorting (FACS) on living cells showed that GPI-anchored TFPI represents about 70–80% of cell surface TFPI. Staining of TFPI on and in fixed and permeabilized cells (total TFPI) demonstrated that GPI-anchored cell surface TFPI contributes to ∼20% of total cellular TFPI. Enzyme-linked immunosorbent assay (ELISA) showed that PI-PLC treatment released a TFPI protein lacking the KD3 and basic C-terminus. These findings strongly suggest that TFPIβ is the predominant GPI-anchored form of TFPI on HUVECs. FX activation assays performed on the cell surface of PI-PLC treated living HUVECs showed the importance of GPI-anchored TFPI on extrinsic Xase complex activity. PI-PLC treatment resulted in increased FX activation. Although GPI-anchored TFPI displays ∼70–80% of cell surface TFPI, overall FXa generation was increased only by ∼50%. In conclusion, HUVEC surface TFPI is predominantly TFPIβ, and GPI-anchored TFPI is the main but not sole regulator of FX activation on the surface of HUVECs. Disclosures: No relevant conflicts of interest to declare.

Factor XII-dependent Contact Activation on Endothelial Cells and Binding Proteins gC1qR and Cytokeratin 1

2001 ◽  
Vol 85 (01) ◽  
pp. 119-124 ◽  
Author(s):  
Kusumam Joseph ◽  
Yoji Shibayama ◽  
Berhane Ghebrehiwet ◽  
Allen Kaplan
Keyword(s):  
Endothelial Cells ◽  
Surface Proteins ◽  
Factor Xii ◽  
Contact Activation ◽  
Endothelial Cell Surface ◽  
Plasma Activation ◽  
Normal Plasma ◽  
Proteolytic Cascade ◽  
Binding Factor ◽  
Do So

SummaryAlthough proteins of the kinin-forming pathway are bound along the surface of endothelial cells, the mechanism of activation of this proteolytic cascade is unclear. Endothelial cell surface proteins, gC1qR and cytokeratin 1, are capable of binding Factor XII and high molecular weight kininogen (HK) in a zinc-dependent reaction thus we considered the possibility that these proteins might catalyze initiation of the cascade. Incubation of Factor XII, prekallikrein, and HK with gC1qR or cytokeratin 1 leads to a zinc-dependent and Factor XII-dependent conversion of prekallikrein to kallikrein. We also demonstrate that normal plasma is capable of activating upon interaction with the cells whereas plasma deficient in Factor XII, prekallikrein and HK do not activate. Normal plasma activation was inhibitable by antibody to gC1qR and cytokeratin 1. Thus, gC1qR and cytokeratin 1, represent potential initiating surfaces for activation of the plasma kinin-forming cascade and may do so as a result of their expression along cell surfaces.

scholarly journals High Molecular Weight Kininogen Regulates Prekallikrein Assembly and Activation on Endothelial Cells: A Novel Mechanism for Contact Activation

Blood ◽  
1998 ◽  
Vol 91 (2) ◽  
pp. 516-528 ◽  
Author(s):  
Guacyara Motta ◽  
Rasmus Rojkjaer ◽  
Ahmed A.K. Hasan ◽  
Douglas B. Cines ◽  
Alvin H. Schmaier
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
High Molecular Weight ◽  
Factor Xii ◽  
Plasminogen Activation ◽  
High Molecular Weight Kininogen ◽  
Contact Activation ◽  
Human Endothelial Cells ◽  
Exogenous Factor

The consequences of assembling the contact system of proteins on the surface of vascular cells has received little study. We asked whether assembly of these proteins on the surface of cultured human endothelial cells (HUVECs) results in the activation of prekallikrein (PK) and its dependent pathways. Biotinylated PK binds specifically and reversibly to HUVECs in the presence of high molecular weight kininogen (HK) (apparent Kd of 23 ± 11 nmol/L,Bmax of 1.7 ± 0.5 × 107 sites per cell [mean ± SD, n = 5 experiments]). Cell-associated PK is rapidly converted to kallikrein. Surprisingly, the activation of cell-associated HK•PK complexes is entirely independent of exogenous factor XII (Km = 30 nmol/L,Vmax = 12 ± 3 pmol/L/min in the absencevKm = 20 nmol/L,Vmax = 9.2 ± 2.1 pmol/L/min in the presence of factor XII). Rather, kallikrein formation is mediated by an endothelial cell-associated, thiol protease. Cell-associated HK is proteolyzed during the course of prekallikrein activation, releasing kallikrein from the surface. Furthermore, activation of PK bound to HK on HUVECs promotes kallikrein-dependent activation of pro-urokinase, resulting in the formation of plasmin. These results indicate the existence of a previously undescribed, factor XII-independent pathway for contact factor activation on HUVECs that regulates the production of bradykinin and may contribute to cell-associated plasminogen activation in vivo.

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

Blood ◽  
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.

scholarly journals An Antibody to Tissue Factor Pathway Inhibitor (PF-06741086) in Combination with Recombinant Factor VIIa Increases Hemostasis in Hemophilia Plasma without Excessive Thrombin Generation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2566-2566 ◽  
Author(s):  
Swapnil Rakhe ◽  
Sunita Patel Hett ◽  
John E. Murphy ◽  
Debra D Pittman
Keyword(s):  
Inhibitory Activity ◽  
Thrombin Generation ◽  
Tissue Factor Pathway Inhibitor ◽  
Hemophilia A ◽  
Recombinant Factor Viia ◽  
Factor Viia ◽  
Lag Time ◽  
Hemophilia B ◽  
Extrinsic Pathway ◽  
Tissue Factor Pathway

Abstract Hemophilia is a hereditary bleeding disorder caused by intrinsic coagulation pathway deficiencies of Factor VIII (hemophilia A) or Factor IX (hemophilia B). Tissue factor pathway inhibitor (TFPI) is a Kunitz-type serine protease inhibitor that negatively regulates thrombin generation within the extrinsic pathway of coagulation. In hemophilia patients the extrinsic pathway remains intact and thus augmentation of this pathway may circumvent the clotting deficiency in hemophilia. PF-06741086, a monoclonal antibody that binds to and neutralizes the inhibitory activity of TFPI is being developed as a potential treatment for bleeding disorders including hemophilia A and hemophilia B with and without inhibitors. Currently, treatment of inhibitor patients is managed by bypass treatments, such as recombinant Factor VIIa (rFVIIa). The effect of PF-06741086 on thrombin generation in the presence of increasing concentrations of rFVIIa (0.0002 to 20 µg/mL) was studied in severe hemophilia A plasma. A dose-dependent increase in thrombin generation was observed over vehicle control with the addition of rFVIIa to the hemophilia plasma. Addition of a fixed concentration of PF-06741086 (16 µg/mL) in combination with rFVIIa resulted in an increase in thrombin generation including higher peak thrombin and shortening of lag time compared to rFVIIa alone. The TGA profiles with the combination of PF-06741086 and rFVIIa at 0.2, 2, and 20 µg/mL were similar suggesting a saturation of mechanism at these concentrations. The combination of PF-06741086 and rFVIIa restored thrombin generation to normal plasma levels at all rFVIIa concentrations examined. The TFPI inhibitory activity of PF-06741086 on thrombin generation in the presence and absence of rFVIIa was further studied in additional hemophilia A plasmas, including hemophilia A plasmas with inhibitors and hemophilia B plasma. All donors had less than 1% coagulation factor activity. A rFVIIa concentration of 2 µg/mL was selected because it corresponded to plasma levels that could be observed following dosing of FVIIa and because the thrombin generation response in hemophilia plasma was similar with FVIIa added to hemophilia A plasma at 0.2, 2 and 20 µg/mL. The concentration of PF-06741086 was 16 µg/mL in these studies. The effect of PF-06741086 on thrombin generation was also measured in non-hemophilic plasma which would have the full complement of coagulation factors. The addition of PF-06741086 alone or in combination with rFVIIa to hemophilia A and B plasma resulted in an increase in thrombin generation including higher peak thrombin concentration and shortening of lag time compared to addition of rFVIIa alone. In hemophilic plasma samples with inhibitors (3 - 1261 Bethesda Units), PF-06741086 alone also restored thrombin generation. A minimal additive effect in peak thrombin generation was observed with the combination of PF-06741086 (16 µg/mL) and 2 µg/mL rFVIII. The midpoint peak thrombin levels achieved with PF-06741086 alone or in combination with rFVIIa were similar to those observed in non-hemophilic plasma and did not exceed the level observed in non-hemophilic plasma dosed with PF-06741086. To summarize, use of rFVIIa in combination with PF-06741086 results in increased thrombin generation in hemophilia A, hemophilia B and inhibitor plasmas without inducing excessive coagulation. Disclosures Rakhe: Pfizer: Employment. Hett:Pfizer: Employment. Murphy:Pfizer: Employment. Pittman:Pfizer: Employment.

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