Biomedical subjects

T Lindhout

Publications and source records attributed to T Lindhout.

At least 19 recordsLinked to original sources

PubMed2006

Anticoagulant and antithrombotic properties of intracellular protease-activated receptor antagonists.

BACKGROUND: Blockade of the thrombin receptors protease-activated receptor (PAR)1 and PAR4 with pepducins, cell-penetrating lipopeptides based on the third intracellular loop of PAR1 and PAR4, effectively inhibits platelet aggregation. We have previously shown that PAR1 pepducin also exerts an anticoagulant activity by partial inhibition of the thrombin plus collagen-induced externalization of phosphatidylserine (PS) at the platelet plasma membrane. OBJECTIVE: In the present study we examined the effects of PAR1 and PAR4 pepducins on tissue factor (TF)-initiated thrombin generation in platelet-rich plasma (PRP) and the interaction between PAR4 pepducin-loaded mouse platelets and a growing thrombus to confirm the relevance of the in vitro data. RESULTS: Localization of pepducins at the inner leaflet of the plasma membrane was confirmed with a fluorescence resonance energy transfer assay. Both the PAR1 pepducin, P1pal12, and the PAR4 pepducin, P4pal10, inhibited TF-initiated thrombin generation in PRP. Concentrations of P1pal12 and P4pal10, which blocked the thrombin-induced influx of extracellular calcium ions and inhibited platelet aggregation, reduced the rate of thrombin generation during the propagation phase by 38% and 36%, respectively. Whether this anticoagulant effect is relevant in inhibiting in vivo arterial thrombin growth is uncertain because P4pal10 prevented the incorporation of platelets in a growing thrombus. CONCLUSIONS: Our findings suggest that in spite of their potential anticoagulant activities the in vivo antithrombotic effect of intracellular PAR pepducins is mainly based on inhibiting platelet-platelet interactions.

Animals↗

PubMed2006

Rolling and adhesion of apoptotic monocytes is impaired by loss of functional cell surface-expressed P-selectin glycoprotein ligand-1.

BACKGROUND: Apoptosis induces cellular membrane changes that are thought to be linked to thrombotic processes, for example, surface exposure of procoagulant phosphatidylserine (PtdSer), upregulation of tissue factor (TF), and microvesicle formation. The latter, though, could downregulate this cellular response by shedding prothrombotic membrane elements, for example, integrins and TF. To test this hypothesis, etoposide-treated, apoptotic, monocytic cells (human monocytic leukemia cell line [THP-1]) were examined for rolling and adhesion on adherent platelets and for TF expression. METHODS AND RESULTS: Etoposide treatment did not result in a significant change in TF antigen expression. However, TF activity, measured in a continuous factor Xa generation assay, was increased fivefold concomitantly with increased exposure of PtdSer. Laminar flow adhesion assays specific for interaction between P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) revealed that in contrast to non-treated cells, apoptotic cells did not roll or firmly attach on adherent platelets. Lack of apoptotic THP-1 platelet interaction could be attributed to both a loss of cell surface-expressed PSGL-1 and loss of functional PSGL-1 as a result of disruption of the binding of PSGL-1 with the cytoskeleton. CONCLUSION: Etoposide-induced apoptosis in THP-1 cells evokes a procoagulant response by increasing TF activity associated with an increased PtdSer exposure. However, in contrast to TF, PSGL-1 shedding and loss of function, makes that apoptotic monocytes are unlikely involved in a thrombotic action because of their inability to adhere to an injured vessel wall or developing thrombus.

Apoptosis↗

PubMed2005

Activated scramblase and inhibited aminophospholipid translocase cause phosphatidylserine exposure in a distinct platelet fraction.

Platelet procoagulant activity is mainly determined by the extent of surface-exposed phosphatidylserine (PS), controlled by the activity of aminophospholipid translocase and phospholipid scramblase. Here, we studied both transport activities in single platelets upon stimulation with various agonists. Besides the formation of procoagulant microparticles, the results show that a distinct fraction of the platelets exposes PS when stimulated. The extent of PS exposure in these platelet fractions was similar to that in platelets challenged with Ca2+-ionophore, where all cells exhibit maximal attainable PS exposure. The size of the PS-exposing fraction depends on the agonist and is proportional to the platelet procoagulant activity. Scramblase activity was observed only in the PS-exposing platelet fraction, whereas translocase activity was exclusively detectable in the fraction that did not expose PS. We conclude that, irrespective of the agonist, procoagulant platelets exhibit maximal surface exposure of PS by switching on scramblase and inhibiting translocase activity.

Annexin A5↗

PubMed2003

von Willebrand factor stimulates thrombin-induced exposure of procoagulant phospholipids on the surface of fibrin-adherent platelets.

Studies from our laboratory have demonstrated that von Willebrand factor (VWF) stimulates thrombin generation in platelet-rich plasma. The precise role of VWF and fibrin in this reaction, however, remained to be clarified. In the present study we utilized thrombin-free planar fibrin layers and washed platelets to examine the relationship between platelet-fibrin interaction and exposure of coagulation-stimulating phosphatidylserine (PS) under conditions of low and high shear stress. Our study confirms that platelet adhesion to fibrin at a shear rate of 1000 s(-1) requires fibrin-bound VWF. The cytosolic calcium concentration ([Ca(2+)]i) of stationary platelets was not elevated and PS exposing platelets were virtually absent (2 +/- 2%). However, thrombin activation resulted in a marked increase in the number of PS exposing platelets (up to 85 +/- 14%) along with a transient elevation in [Ca(2+)]i from 0.05 micro mol L(-1) up to 1.1 +/- 0.2 micro mol L(-1). Platelet adhesion to fibrin at a shear rate of 50 s(-1) is mediated by thrombin but not by fibrin-bound VWF. The [Ca(2+)]i of these thrombin-activated platelets was elevated (0.2 +/- 0.1 micro mol L(-1)), but only a minority of the platelets (11 +/- 8%) exposed PS. The essential role of VWF in this thrombin-induced procoagulant response became apparent from low shear rate perfusion studies over fibrin that was incubated with VWF and botrocetin. After treatment with thrombin, the majority of the adherent platelets (57 +/- 23%) exposed PS and had peak values of [Ca(2+)]i of about 0.6 micro mol L(-1). Taken together, these results demonstrate that thrombin-induced exposure of PS and high calcium response on fibrin-adherent platelets depends on shear- or botrocetin-induced VWF-platelet interaction.

Blood Coagulation↗

PubMed2002

Exposure of procoagulant phospholipids on the surface of platelets in patients undergoing cardiopulmonary bypass using non-coated and heparin-coated extracorporeal circuits.

Cardiopulmonary bypass (CPB) is associated with a generalized hemostatic defect, in which platelet dysfunction seems to play a central role. The present study was designed to elucidate whether the potential procoagulant activity of platelets, detected as annexin V binding, was altered during coronary bypass surgery, using non-coated and heparin-coated extracorporeal circuits. Thirty patients undergoing elective coronary artery bypass grafting were prospectively randomized using either a standard untreated extracorporeal circuit (n = 15) or a heparin-treated extracorporeal circuit (n=15). Besides measurement of the procoagulant phospholipid activity, the mediastinal blood loss after surgery, and the blood transfusion requirements were also monitored. CPB induced a decrease in the percentage of activated platelets in whole blood, manifest directly after start of CPB, which was significantly attenuated using a non-treated system. Postoperatively, the percentage of activated platelets recovered in both systems, reaching a point of significance 24 hours after the operation, compared to the values 2 hours after the operation. The differences among the groups for mediastinal blood loss during the first 2 and 24 postoperative hours coincided with the differences in procoagulant phospholipid activity. Furthermore, there was no statistical difference among the groups for blood transfusion requirements. The platelets in both groups showed a significantly lower ability to generate ionomycin-induced procoagulant activity after blood-material interaction when compared to the baseline values. These observations are compatible with the notion that during CPB, irrespective of the heparin coating, platelets become modestly activated.

Aged↗

PubMed2001

Glycoprotein VI but not alpha2beta1 integrin is essential for platelet interaction with collagen.

Platelet adhesion on and activation by components of the extracellular matrix are crucial to arrest post-traumatic bleeding, but can also harm tissue by occluding diseased vessels. Integrin alpha2beta1 is thought to be essential for platelet adhesion to subendothelial collagens, facilitating subsequent interactions with the activating platelet collagen receptor, glycoprotein VI (GPVI). Here we show that Cre/loxP-mediated loss of beta1 integrin on platelets has no significant effect on the bleeding time in mice. Aggregation of beta1-null platelets to native fibrillar collagen is delayed, but not reduced, whereas aggregation to enzymatically digested soluble collagen is abolished. Furthermore, beta1-null platelets adhere to fibrillar, but not soluble collagen under static as well as low (150 s(-1)) and high (1000 s(-1)) shear flow conditions, probably through binding of alphaIIbbeta3 to von Willebrand factor. On the other hand, we show that platelets lacking GPVI can not activate integrins and consequently fail to adhere to and aggregate on fibrillar as well as soluble collagen. These data show that GPVI plays the central role in platelet-collagen interactions by activating different adhesive receptors, including alpha2beta1 integrin, which strengthens adhesion without being essential.

Adenosine Diphosphate↗

PubMed2001

Tissue factor activity in human monocytes is regulated by plasma: implications for the high and low responder phenomenon.

The 'high and low responder phenomenon' of monocyte tissue factor (MTF) activity has been attributed to effects on monocytes by granulocytes, platelets and lipopolysaccharide (LPS). To study the possible contribution of plasma to the high and low responder phenomenon, we measured the MTF activity in isolated cryopreserved human monocytes from two donors (monocytes A and monocytes B) after incubation in a plasma environment depleted of granulocytes, platelets and LPS. In buffer only, MTF activity was 643 and 679 fM (fM = final concentration of tissue factor), in normal pooled plasma, it was 1478 and 1615 fM (P = 0.001), respectively, in monocytes A and in monocytes B. Incubation with individual plasma samples from healthy controls (n = 43) gave a median MTF of 1355 fM (range 1044-1976 fM) and 1329 fM (range 858-1951 fM) respectively. A plasma consistently induced a higher or lower level of MTF activity in both monocytes: r = 0.82 (P < 0.00001). Coumarin use did not influence the high and low responder phenomenon. In the absence of granulocytes, platelets and LPS, plasma determines the high and low responder phenomenon. This phenomenon is not influenced by coumarin treatment.

Aged↗

PubMed2001

Contribution of platelet-derived factor Va to thrombin generation on immobilized collagen- and fibrinogen-adherent platelets.

Adhesion of platelets to immobilized collagen induces the expression of anionic phospholipids, e.g. phosphatidylserine (PS), in the outer leaflet of the plasma membrane of these platelets. In contrast, of the platelets that adhere to immobilized fibrinogen only a small sub-population representing 10 +/- 3% of the total population of the fibrinogen-adherent platelets has exposed PS as probed by annexin V binding. Although the presence of PS is thought to be critical for thrombin generation at the platelet surface, no information is available about the effect of this differential PS exposure on the ability of adherent platelets to support thrombin generation. Perfusion of the fibrinogen- or collagen-adherent platelets with solutions containing factor Xa and prothrombin resulted in thrombin generation that i) increased linear during the first perfusion minutes, ii) was about two-fold faster at collagen-adherent than at fibrinogen-adherent platelets and iii) was for more than 98% restricted to the surface of the adherent platelets. It appeared that the lower thrombin generating capacity of fibrinogen-adherent platelets is not due to a lower overall surface density of PS, but is caused by lower amounts of platelet-bound factor Va. Firstly, in both cases thrombin generation could be completely attenuated with antibodies against human factor Va, and secondly, in the presence of an excess of exogenous plasma-derived factor Va similar initial rates of thrombin formation were measured for collagen- and fibrinogen-adherent platelets. Our findings suggest a unique role for immobilized collagen in maintaining haemostasis.

Blood Platelets↗

PubMed2000

Regulation of platelet factor Va-dependent thrombin generation by activated protein C at the surface of collagen-adherent platelets.

Recent studies have indicated that factor Va bound to activated platelets is partially protected from inactivation by activated protein C (APC). To explore whether this sustained factor Va activity could maintain ongoing thrombin generation, the kinetics of platelet factor Va-dependent prothrombinase activity and its inhibition by APC were studied. In an attempt to mimic physiologically relevant conditions, platelets were adhered to collagen type I-coated discs. These discs were then spun in solutions containing prothrombin and factor Xa either in the absence or presence of APC. The experiments were performed in the absence of platelet-derived microparticles, with thrombin generation and inhibition confined to the surface of the adherent platelets. APC completely inactivated platelet-associated prothrombinase activity with an overall second order rate constant of 3.3 x 10(6) m(-)1 s(-)1, which was independent of the prothrombin concentration over a wide range around the apparent K(m) for prothrombin. Kinetic studies on prothrombinase assembled at a planar phospholipid membrane composed of 25 mol % phosphatidylserine and 75 mol % phosphatidylcholine revealed a similar second order rate constant of inhibition (2.5 x 10(6) m(-1) s(-1)). Collectively, these data demonstrate that ongoing platelet factor Va-dependent thrombin generation at the surface of collagen-adherent platelets is effectively inhibited by APC. No differences were observed between the kinetics of APC inactivation of plasma-derived factor Va or platelet factor Va as part of the prothrombinase associated with, respectively, a planar membrane of synthetic phospholipids or collagen-adherent platelets.

Anticoagulants↗

PubMed2000

Receptors and signalling mechanisms in the procoagulant response of platelets.

Platelets in an advanced stage of activation change from coagulation-inactive to coagulation-promoting cells. This procoagulant response is characterised by exposure of aminophospholipids, such as phosphatidylserine, to the platelet surface and by formation of microvesicles. Under specific conditions, when both signalling and adhesive platelet receptors are occupied, collagen and also thrombin are able to trigger this response. Thus, platelets express high coagulation-promoting activity only after interacting with multiple receptors.

Blood Coagulation↗

PubMed2000

Antibodies to beta2-glycoprotein I associated with antiphospholipid syndrome suppress the inhibitory activity of tissue factor pathway inhibitor.

Anionic phospholipid membranes have a dual role in blood coagulation: they are essential for the initiation and propagation as well as for the limitation and termination of the blood coagulation process. Patients with the anti-phospholipid syndrome (APS) carrying antibodies against complexes of anionic phospholipids and plasma proteins, show in vitro inhibited phospholipid dependent coagulation reactions, whereas in vivo the presence of these antibodies is associated with an increased risk of thrombosis. In this study we focussed on the effects of these anti-phospholipid antibodies on the regulation of TF-mediated factor Xa (FXa) generation in plasma. We hypothesized that anti-phospholipid antibodies interfere with the phospholipid-dependent inhibition by tissue factor pathway inhibitor (TFPI) of TF-induced coagulation. Indeed, total-IgG, anti-cardiolipin-IgG (aCL) and anti-beta2GPI-IgG, isolated from patient plasmas, all stimulated TF-induced FXa generation in normal plasma. This enhanced FXa generation was not observed when the patient's IgG was depleted of anti-beta2GPI-IgG or when normal plasma was depleted of beta2PGPI or TFPI. Taken together, these data indicate that antibodies to beta2GPI, circulating in patients with APS, suppress TFPI-dependent inhibition of TF-induced coagulation, which results in an increased FXa generation.

Antiphospholipid Syndrome↗

PubMed1999

Inhibition of tissue factor-factor VIIa-catalyzed factor X activation by factor Xa-tissue factor pathway inhibitor. A rotating disc study on the effect of phospholipid membrane composition.

The physiological inhibitor of tissue factor (TF).factor VIIa (FVIIa), full-length tissue factor pathway inhibitor (TFPI(FL)) in complex with factor Xa (FXa), has a high affinity for anionic phospholipid membranes. The role of anionic phospholipids in the inhibition of TF.FVIIa-catalyzed FX activation was investigated. FXa generation at a rotating disc coated with TF embedded in a membrane composed of pure phosphatidylcholine (TF.PC) or 25% phosphatidylserine and 75% phosphatidylcholine (TF.PSPC) was measured in the presence of preformed complexes of FXa.TFPI(FL) or FXa.TFPI(1-161) (TFPI lacking the third Kunitz domain and C terminus). At TF.PC, FXa.TFPI(FL) and FXa.TFPI(1-161) showed similar rate constants of inhibition (0.07 x 10(8) M(-1) s(-1) and 0.1 x 10(8) M(-1) s(-1), respectively). With phosphatidylserine present, the rate constant of inhibition for FXa.TFPI(FL) increased 3-fold compared with a 9-fold increase in the rate constant for FXa. TFPI(1-161). Incubation of TF.PSPC with FXa.TFPI(FL) in the absence of FVIIa followed by depletion of solution FXa.TFPI(FL) showed that FXa.TFPI(FL) remained bound at the membrane and pursued its inhibitory activity. This was not observed with FXa.TFPI(1-161) or at TF.PC membranes. These data suggest that the membrane-bound pool of FXa.TFPI(FL) may be of physiological importance in an on-site regulation of TF.FVIIa activity.

Catalysis↗

PubMed1999

Heterogeneity in microparticle formation and exposure of anionic phospholipids at the plasma membrane of single adherent platelets.

Adherent platelets were examined for their ability to form microvesicles and procoagulant sites for thrombin formation. Epifluorescence and phase-contrast microscopy were employed to visualize shape changes, changes in intracellular Ca(2+) levels ([Ca(2+)](i)), vesiculation of the plasma membrane and appearance of anionic phospholipids in the outer leaflet of the plasma membrane, as probed by annexin V binding. In the absence of extracellular Ca(2+) two stable populations of adherent platelets were observed. The majority of the adherent platelets were fully spread and about 10% remained in a non-spread dendritic state. In the presence of extracellular Ca(2+) vesiculation at the surface of spread platelets occurred at a rather slow rate (10% of the platelets after 20 min) concomitantly with an increase in [Ca(2+)](i) and binding of annexin V. However, a small fraction of the adherent platelets ( approximately 1%) responded much faster. Ionomycin-enhanced influx of Ca(2+) in dendritic platelets resulted in a rapid transformation of these platelets into inflated, balloon-shaped, platelets having a diameter of 2.0+/-0.7 microm without notable microvesicle formation. In contrast, fully spread platelets retained their shape but obtained frayed edges as a result of microvesicle formation. Confocal scanning fluorescence microscopy indicated that annexin V bound to very distinct sites at the outer plasma membrane of spread as well as balloon-shaped platelets. Inhibition of platelet calpain activity suppressed ionomycin-enhanced microvesicle formation and ballooning of platelets, but not annexin V binding. These findings indicate that vesiculation and ballooning, but not the exposure of phosphatidylserine at the outer leaflet of the adherent platelet membrane, are associated with cytoskeleton destruction. Altogether, the data suggest a similar relationship between [Ca(2+)](i) and the formation of platelet procoagulant sites as reported for platelets in suspension. However, the present investigations on single adherent platelets reveal for the first time that adhesion and spreading of platelets is not necessarily associated with the appearance of procoagulant sites. Secondly, an unexpected diversity was observed among adherent platelets with respect to sensitivity to Ca(2+)-induced generation of procoagulant sites and Ca(2+)-induced vesiculation of plasma membrane. It is tempting to speculate that this diversity is of importance for the procoagulant response of platelets to a hemostatic challenge elicited by an injured vessel wall.

Anions↗

PubMed1999

Metallic wires with an adherent lubricious and blood-compatible polymeric coating and their use in the manufacture of novel slippery-when-wet guidewires: possible applications related to controlled local drug delivery.

A new procedure was developed for the controlled application of adherent hydrophilic and biocompatible coatings onto the surface of "endless" metallic wires. Use of copolymers of 1-vinyl-2-pyrrolidinone and alkylmethacrylates provided coatings with excellent adherence and lubricity, and markedly low thrombogenicity. Coated wires could be spiralized without damaging the coating; the resulting coils are potentially useful as lubricious guidewires for use in, for example, interventional cardiology or urology. This study demonstrates that the lubricity of the coating is dependent on the composition (hydrophilicity) of the coating biomaterial, as well as on the thickness of the coating. Furthermore, the results imply that the adherence of the hydrophilic coating is essentially due to entanglement of the binder polymer chains and the hydrophilic copolymer chains. Moreover, the idea to use the hydrophilic coating on the wire as a temporary depot for controlled local drug delivery was explored. The coating was loaded with the dye rhodamine, and release of the dye upon immersion of the coated wire in water was studied. This work revealed that release of the drug is dependent on the composition of the coating. The potential utility of such wires with a drug-charged coating for controlled local drug delivery is discussed briefly.

Blood↗

PubMed1998

Transient high affinity binding of tissue factor pathway inhibitor-factor Xa complexes to negatively charged phospholipid membranes.

The interaction of tissue factor pathway inhibitor (TFPI), factor Xa, and TFPI-factor Xa complexes with negatively charged phospholipid membranes composed of 25 mol % phosphatidylserine and 75 mol % phosphatidylcholine was studied by ellipsometry. The binding of TFPI alone was negligible; factor Xa bound with moderate affinity, with a dissociation constant Kd = 42 nM. Formation of the TFPI-factor Xa complex drastically enhanced the affinity for phospholipid membranes, Kd = 5 nM, compared to that of either protein alone. TFPI1-161, a TFPI variant lacking the third Kunitz domain and the positively charged C-terminus did not enhance binding affinity of the factor Xa. Analysis of the kinetics of adsorption and desorption confirmed the equilibrium binding data, although upon longer residence at the lipid membrane the desorption rate of TFPI-factor Xa complexes became slower, indicating an increase in affinity with longer residence of the TFPI-factor Xa complexes at the membrane. In contrast, binding of TFPI-factor Xa complexes in the presence of an excess factor Xa was transient; maximal binding is followed by a slow desorption of the complex. Immunoblot analysis revealed that this desorption was accompanied with cleavage of TFPI by membrane-bound factor Xa. Collectively, our results show that phosphatidylserine containing membranes will accumulate tightly bound TFPI-factor Xa complexes, and that uncomplexed, phospholipid-bound, factor Xa, will cause limited proteolysis of TFPI accompanied by simultaneous release of these complexes from the phospholipid membrane.

Adsorption↗

PubMed1998

Adsorption of proteins onto poly(ether urethane) with a phosphorylcholine moiety and influence of preadsorbed phospholipid.

In a previous report we demonstrated that the blood compatibility of poly(ether urethane) (PEU) was improved by grafting phosphorylcholine (PC) groups on the surface. The improved blood compatibility was indicated by decreased platelet adsorption/activation and reduced thrombin formation at the polymer surface in experiments in which the surfaces were contacted with platelet-rich plasma in vitro. In the present study, we investigated the effect of grafted PC groups at a PEU surface on protein and phospholipid adsorption. Adsorption of human fibrinogen (Fg), human serum albumin (Alb), human high-molecular-weight kininogen (HMWK), and dioleoyl phosphatidylcholine (DOPC) vesicles was measured by ellipsometry. For this purpose, thin PEU films were cast on silicon wafers. The polymer film was photochemically modified with a PC-containing aryl azide. The presence of PC groups on the polymer surface was demonstrated by ESCA (Electron Spectroscopy for Chemical Analysis). The hydrophilicity of the polymer surface increased by the surface modification, as indicated by a decrease of the contact angle from 59 degrees before to 43 degrees after modification. Our data show that the presence of PC groups has little effect on the adsorption of proteins to a PEU surface. The highest adsorption was observed for Fg (0.49 microgram/cm2 on PC-modified PEU and 0.50 microgram/cm2 on PEU), followed by HMWK (0.28 microgram/cm2 on both PC-modified PEU and PEU), and Alb (0.16 microgram/cm2 on PC-modified PEU and 0.18 microgram/cm2 on PEU). Protein adsorption was further studied on a "biomembrane-like" DOPC bilayer formed on hydrophilic silicon. We found no protein adsorption on this DOPC bilayer. The adsorption of small unilamellar DOPC vesicles on the polymer surfaces amounted to about 0.06 microgram/cm2 (corresponding to circa 30% of monolayer coverage) and was similar for PC-modified PEU and PEU. Despite this partial surface coverage, preadsorbed DOPC on the polymer surface diminished the subsequent adsorption of proteins considerably. These results show that the mere presence of phosphorylcholine groups on a PEU surface is insufficient to suppress protein adsorption. The highly ordered structure of natural phospholipid bilayers seems to be required to suppress protein adsorption effectively.

Adsorption↗

PubMed1998

Heparin coating of tantalum coronary stents reduces surface thrombin generation but not factor IXa generation.

In the present study we used an in-vitro technique to examine initiation and propagation of blood coagulation at the surface of tantalum coronary stents exposed to flowing platelet-rich and platelet-free plasma. The time course of factor IXa production at the surface of the stent was not influenced by platelets. In spite of a significant factor IXa production, no thrombin activity was detected when the tantalum stent was exposed to platelet-free plasma; only when the stent was exposed to platelet-rich plasma was extensive thrombin production observed. These findings indicate that tantalum triggers blood coagulation, but that (adherent) platelets are essential for thrombin generation. Heparin-coated tantalum stents exposed to flowing platelet-rich plasma showed that factor IXa generation was slightly reduced compared with the bare stent. However, the heparin coating drastically delayed the onset of thrombin generation and largely reduced the steady-state production of thrombin. We found a clear relationship between the antithrombin binding capacity and the antithrombogenic potential of the heparin-coated stents. The mode of action of immobilized heparin is thought to abrogate thrombin generation by inhibiting thrombin-dependent positive feedback reactions at the surface of the coronary stent.

Animals↗

PubMed1998

Initiation and propagation of blood coagulation at artificial surfaces studied in a capillary flow reactor.

We have made use of a novel flow reactor to study the initiation and propagation of the ex vivo blood coagulation processes at artificial surfaces. The flow reactor consisted of a primary glass or polymer capillary that is connected to a secondary glass capillary, which inner wall was coated with a phospholipid bilayer of 25 mol% dioleoylphosphatidylserine/75 mol% dioleoylphosphatidylcholine (DOPS/DOPC). Citrated platelet free plasma and a CaCl2 solution were delivered by syringe pumps and mixed just before the entrance of the flow reactor. The outflowing plasma was assayed for factor XIa, factor IXa, factor Xa and thrombin activity. Perfusion of recalcified plasma through a bare glass capillary resulted in a transient generation of fluid phase factor XIa. In contrast, factor IXa production increased slowly to attain a stable steady-state level. We established that surface-bound factor XIa was responsible for a continuous production of factor IXa. Factor IXa-induced generation of factor Xa and thrombin was only observed when contact activated plasma was subsequently perfused through a DOPS/DOPC-coated capillary, showing that propagation of the factor IXa trigger requires a procoagulant, phosphatidylserine-containing, phospholipid membrane. The negatively charged inner surface of a heparin-coated polyurethane capillary, generated like the glass capillary significant amounts of factor XIa and factor IXa when perfused with recalcified plasma. No differences were found between unfractionated heparin and heparin devoid of anticoagulant activity. Thus, it is concluded that contact activation and factor IXa generation in flowing plasma is not inhibited by immobilised anticoagulant active heparin. Consequently, factor IXa-dependent thrombin generation at a downstream located phospholipid membrane was similar, regardless the specific anticoagulant activity of immobilised heparin.

Blood Coagulation↗