Improvement of patient education and information: development of a patient's information dossier.

The demand for patient information in modern medical care is increasing and sound information for patients is becoming a necessity. For haemophilia patients, information about their disease and its complications is already widely available. In order to increase the organization of this information, a 'Patient Information Dossier' (PID) and communication check lists were developed at the Dutch National Hemophilia Center, the Van Creveldkliniek, in cooperation with the Department of Patient Education of the University Medical Center Utrecht. The PID has an unique double function: (1) it contains patient tailored information about the practical facts of hospital care; and (2) it provides a communication checklist used by various members of the comprehensive care team, in order to supply patients with more uniform information. In order to gain a better insight of the gaps in information supply, according to patients and healthcare workers, the Department of Patient Education formulated a questionnaire. The PID itself was written by a study group consisting of members of the comprehensive care team. The entire process was developed, edited and coordinated by an advisor of the Department of Patient Education. The above-mentioned study group developed a specific PID on haemophilia care. Its 10 chapters provide information and guidelines, and advise patients where to find more information about this subject. Each chapter includes a checklist for patients, enabling them to prepare subjects for discussion during clinical visits. The team also developed a communication checklist to be used by various team members during a patient's visit to the clinic, as well as specific checklists covering the possible problem subjects of the PID. The PID is the lifelong property of the patient, and can be used during each visit to the clinic. The PID was implemented in February 2000, and within 4 months, was distributed among 200 patients visiting the Van Creveldkliniek. Evaluation by use of a questionnaire showed that most patients found the information in the PID sufficient and in accordance with that which they had received previously.

Protamine sulphate inhibits platelet membrane glycoprotein Ib-von Willebrand factor activity.

Platelet adhesion to the injured vessel wall is essential in haemostasis and thrombosis. This process involves the interaction of the platelet glycoprotein Ib (GPIb) with surface bound von Willebrand factor (vWF). Since synthetic polycationic peptides of the general formula (Arg)n, (Lys)n or (Arg-Lys)n inhibit GPIb-vWF interaction, they were suggested as potential antithrombotics. Protamine sulphate is a highly cationic polypeptide, arginine accounting for approximately 60% of the primary sequence, utilized to neutralize the anticoagulant effect of heparin after cardiac surgery. We have investigated potential effects of protamine sulphate on the function of GPIb-vWF. Addition of protamine sulphate to platelet-rich plasma (PRP), reduced significantly the GPIb-vWF activity as assessed by ristocetin-induced platelet agglutination. When protamine sulphate was added to PRP containing heparin, even at clinically relevant neutralizing doses the GPIb-vWF activity was reduced by 20-30% (p < 0.001). Protamine sulphate in excess of heparin nearly abolished the activity. Furthermore, the direct effect of protamine sulphate on collagen-induced platelet thrombus formation in non-anticoagulated human blood was investigated by employing an ex-vivo parallel-plate perfusion chamber device. Protamine sulphate (200 microg/mL) reduced platelet-collagen adhesion at shear rates of 650 and 2600 sec(-1) by 40% (p< 0.004) and 45% (p < 0.0001), respectively. The corresponding platelet thrombus volumes were concomitantly reduced by 90% (p < 0.006) and 84% (p < 0.05). Our data are questioning the rationale for empirical repetitive protamine sulphate administration when so-called "heparin rebound" after cardiac surgery is suspected, since protamine sulphate in excess of heparin may impair the platelet GPIb-vWF interaction necessary for normal haemostasis.

Loss of ELISA specificity due to biotinylation of monoclonal antibodies.

A significant degree of nonspecificity was found in ELISA determinations of soluble urokinase receptor (suPAR) in human blood plasma when biotinylated monoclonal antibodies (Mabs) were used for the detection layer. Surface plasmon resonance studies using both nonbiotinylated and biotinylated antibodies demonstrated that biotinylation reduced specific binding of the antibodies to their target antigen, suPAR. Furthermore, biotinylation produced a new interaction with unknown human plasma protein(s), unrelated to suPAR. Nonspecific interaction with plasma protein(s) was also observed after biotinylation of a Mab having no specific target antigen in human plasma and, in both cases, the level of nonspecific interaction was directly related to the degree of antibody biotinylation. These results reinforce earlier observations that biotinylation of antibodies can reduce the affinity of antibodies, but also indicate that, in addition, biotinylation can reduce the specificity of immunoassays for plasma proteins.

Soluble urokinase receptor released from human carcinoma cells: a plasma parameter for xenograft tumour studies.

The urokinase plasminogen activator receptor (uPAR) plays a critical role in urokinase-mediated plasminogen activation and thereby in the process leading to invasion and metastasis. Soluble urokinase receptor (suPAR) is released from tumours, and in cancer patients the blood level of soluble receptor is increased. Using an enzyme-linked, immunosorbent assay (ELISA)-specific for the human urokinase receptor, release of soluble receptor was measured in cultures of human breast carcinoma cells, in tumour extracts and in plasma from mice with xenografted human tumours. Soluble human urokinase receptor (shuPAR) was released into culture supernatant during the growth of the human breast cancer cell line MDA-MB-231 BAG, and the level of shuPAR in conditioned medium determined by ELISA was a linear function of both viable cell number and time of incubation. Western blotting showed that the form of shuPAR measured by ELISA in conditioned medium consisted virtually exclusively of the three-domain full-length protein, while uPAR in cell lysates consisted of full-length uPAR as well as the domains (2+3) cleavage product. shuPAR was also released into the plasma of nude mice during growth of MDA-MB-231 BAG, MDA-MB-435 BAG and HCT 116 cells as subcutaneously xenografted tumours. Western blotting demonstrated that the shuPAR released from the xenografted human tumours into plasma consisted of the three-domain full-length protein, despite the finding of some cleaved uPAR in detergent extracts of tumour tissue. The levels of shuPAR determined by ELISA in the plasma of host mice during the growth of xenografted cell lines were highly correlated with tumour volume.

Iohexol, platelet activation and thrombosis. I. Iohexol-induced platelet secretion does not affect thrombus formation in native blood.

BACKGROUND: The nonionic monomer iohexol triggers in vitro platelet secretion of beta-thromboglobulin (beta-TG). This iohexol platelet activation may promote intravascular thrombosis. We studied this relationship by employing a human model of collagen-induced platelet thrombus formation at arterial flow. The ionic dimer ioxaglate, the nonionic dimer iodixanol, and glucose were included. METHODS AND RESULTS: In vitro platelet activation as measured by beta-TG secretion following a 1-min incubation of native blood with 50 vol% of iohexol was significant. Glucose solutions of 300, 580 and 825 mosmol, corresponding to the osmolalities of respectively iodixanol, ioxaglate and iohexol, increased the beta-TG secretion in parallel with the osmolalities. Ioxaglate and iodixanol were virtually inert. Continuous infusion of iohexol or 580 or 825 mosmol glucose (40 vol%) into flowing native blood at an arterial wall shear rate of 2600 s-1 in an ex vivo collagen-induced platelet thrombus formation device triggered pronounced secretion of beta-TG. However, the platelet thrombus formation in blood mixed with iohexol was within the same range as that observed with ioxaglate or iodixanol. Increasing glucose osmolality induced increasing beta-TG secretion, which paralleled gradually decreasing platelet thrombus formation. CONCLUSION: Iohexol and 580 or 825 mosmol glucose trigger platelet secretion of beta-TG. This secretion is not associated with enhanced collagen-induced platelet thrombus formation at high arterial shear.

Iohexol, platelet activation and thrombosis. II. Iohexol-induced platelet secretion does not affect collagen-induced or tissue-factor-induced thrombus formation in blood that is anticoagulated with heparin and aspirin.

BACKGROUND: There is a dispute about the potential effects of radiographic contrast media (CM) on thrombogenesis. The nonionic CM iohexol triggers platelet beta-thromboglobulin (beta-TG) secretion, and thus may activate the platelets and promote thrombosis. We addressed this topic in a study employing a human model of arterial thrombus formation in the presence of aspirin and heparin. This was a follow-up to our initial study (on thrombus formation in native blood) which did not include antithrombotic drugs. The nonionic CM iohexol (monomer) and iodixanol (dimer) and the ionic CM ioxaglate (dimer) were compared. METHODS AND RESULTS: Thrombus formation was triggered by a surface rich in either collagen or tissue factor, positioned in a parallel-plate perfusion chamber device at an arterial wall shear rate of 2600 s-1. Blood from healthy volunteers, following ingestion of 1 g aspirin, was mixed with 40 vol% CM and 2.0 IU/ml heparin and passed over the surfaces. Thrombus formation in the presence of either CM showed no difference, despite the fact that iohexol triggered a pronounced platelet beta-TG secretion; iodixanol or ioxaglate were virtually inert. CONCLUSION: There was no association between iohexol-induced beta-TG secretion and thrombus formation on collagen (platelet-driven) or on tissue factor (thrombin-driven) in the presence of a standard antithrombotic regimen of aspirin and heparin as used in the clinic. The notion of a thrombotic risk due to platelet activation by iohexol was thus not substantiated by this study.

Monocyte procoagulant activity induced by adherence to an artificial surface is reduced by end-point immobilized heparin-coating of the surface.

Heparin-coating improves the biocompatibility of blood contacting artificial surfaces. This led us to investigate the impact of heparin-coating (Carmeda AB, Stockholm) of polymetylmetacrylate on the expression of monocyte tissue factor procoagulant activity (TF-PCA) by surface adhesion. Also, the anticoagulant effect of heparin-coating in the presence or absence of adherent procoagulant monocytes was assessed. This is of particular interest, since activation of extrinsic coagulation by adherent monocyte TF-PCA may play a significant role in thrombin generation during extracorporeal circulation. Monocytes exposed to heparin-coated or non-coated polymetylmetacrylate expressed TF-PCA. The heparin coat did not affect the rate of monocyte adhesion. However, heparin-coating reduced the induction of TF-PCA of non-adherent and adherent monocytes by 17 and 33% (p <0.001 and p <0.0003), respectively. Heparin-coating in the absence of monocytes, totally inhibited the clotting of recalcified plasma (p <0.003). In contrast, in the presence of adherent monocytes expressing TF-PCA, surface-bound heparin did not inhibit clotting. However, inclusion of heparin in a plasma concentration of 8.9 IU/ml totally inhibited the activation of coagulation. It is apparent that heparin-coating of an artificial surface is an efficient means to inhibit coagulation of recalcified plasma, but much less so when procoagulant monocytes are adherent to the coated surface. The present findings are of clinical relevance, since monocytes will adhere to blood contacting surfaces of extracorporeal circuits or to implanted vascular prostheses and subsequently express TF-PCA, and this may promote thromboembolism.

ELISA determination of soluble urokinase receptor in blood from healthy donors and cancer patients.

Measurement of urokinase receptor (uPAR) in tumor extracts has prognostic value, but assay of the soluble uPAR (suPAR) in peripheral blood may offer wider applications in cancer patient management. A tumor extract uPAR ELISA was modified to eliminate nonspecific plasma protein interference, enabling specific detection of suPAR in plasma and sera with >90% recovery of added calibrator. suPAR concentrations in citrate plasma correlated with sera in 93 healthy blood donors (r = 0.84, P <0.0001), with a median value for both of 1.2 microg/L. The plasma median for 19 advanced breast cancer patients was 2.9 microg/L suPAR, and a similar increase was found for 10 advanced colon cancer patients, consistent with release of suPAR from tumors into blood. Repetitive monitoring of suPAR in cancer patients' blood may have value in assessment of prognosis and tumor recurrence.

A peptide sequence from the EGF-2 like domain of FVII inhibits TF-dependent FX activation.

We have found that synthetic peptides derived from the two epidermal growth factor-like domains of factor VII are inhibitors of tissue factor dependent factor X activation. Inhibition was most pronounced for a constrained sequence of amino acids corresponding to positions 91-102 of factor VII, Cys-Val-Asn-Glu-Asn-Gly-Gly-Cys-Glu-Gin-Tyr-Cys. The biological activity appeared to be localized to the tripeptide 'motif', Glu-Gln-Tyr, within the larger sequence. The cyclic peptide was also an inhibitor of tissue factor induced coagulation of plasma, using lipidated tissue factor or tissue factor expressed on the surface of living cells. However, it did not interfere with intrinsic coagulation. Inhibition of factor X activation was dose-dependent with an IC50 value of 350 microM. Kinetic analyses revealed non-competitive inhibition with respect to factor X and suggested that the peptide sequence interferes with the factor VII/tissue factor/factor X complex formation and function. A pentapeptide analog of the putative pharmacophore was also a dose-dependent inhibitor of factor X activation with an IC50 value of 560 microM, but the tripeptide, Glu-Gin-Tyr, alone was without effect. Our results suggest a direct role for the second epidermal growth factor-like domain of factor VII, and in particular its loop I, in the formation and function of the factor VII/tissue factor/factor X complex.

Shear-induced platelet activation and platelet microparticle formation at blood flow conditions as in arteries with a severe stenosis.

In the present study, we investigated whether high arterial shear stresses at various exposure times or a sudden increase in shear stress introduced by a stenosis affect platelet activation and platelet microparticle formation in native human blood. We used a parallel-plate perfusion chamber device through which nonanticoagulated human blood was drawn (10 mL/min) by a pump directly from an antecubital vein through the flow channel of a perfusion chamber at wall shear rates of 420, 2600, and 10500 s-1. In another set of experiments, an eccentric stenosis was introduced into the flow channel. Wall shear rates of 2600 or 10500 s-1 at the stenosis apex were maintained at the same flow rate. The wall shear rate upstream and downstream of these stenoses was 420 s-1. A shear rate of 420 s-1 is within the range of those encountered in healthy small coronary arteries, whereas those of 2600 and 10500 s-1 are representative for vessels with various degrees of stenotic lesions. The blood was exposed to these shear rates for periods varying from 0.075 to 3.045 seconds. Platelet activation was assessed as activated glycoprotein (GP) IIb/IIIa by FITC-labeled monoclonal antibody (MAb) PAC-1 and aminophospholipid translocation by FITC-labeled annexin V. Microparticle formation was quantified by FITC-labeled MAb Y2/51 directed against GP IIIa. Significant platelet activation and formation of microparticles were observed at 10500 s-1 only (P < .008). This shear-induced platelet activation and microparticle formation were enhanced by introduction of a thrombus-promoting surface consisting of type III human collagen fibrils. Introduction of the most severe stenosis at 10500 s-1 further increased platelet activation (P < .017). The collagen-induced thrombus formation increased the platelet thrombus volume at 10500 s-1 from 16.5 to 33.8 microns3/microns2 (P < .003) on the stenosis apex when the most severe stenosis was used. A correlation (P < .0001) between platelet thrombus volume and platelet microparticle formation was observed in the presence of the eccentric stenoses. Apparently, high shear stress (315 dynes/cm2 at 10500 s-1), as encountered in severe atherosclerotic arteries, activated platelets and triggered platelet microparticle formation. In contrast, no significant platelet activation or formation of platelet microparticles was observed at physiological shear (420 s-1) or at the shear condition simulating shear in arteries with a less severe stenosis (2600 s-1). The data imply that platelets are activated and form microparticles in native blood at very high shear stresses. These events are potentiated by prolonged exposure to the high shear or by a sudden change of increasing shear due to the stenosis. The latter situation apparently enhances platelet thrombus formation at the stenosis.

Characterisation of cell-surface procoagulant activities using a microcarrier model.

A novel model is described for characterisation of cell-surface procoagulant activities and their inhibitors. Microcarrier beads were used to present living cells to recalcified blood plasma in the stirred measuring wells of an electromagnetic coagulometer. By this means the procoagulant activity on the surface of the cells could be automatically determined as clotting time. Procoagulant activity was investigated on normal and transformed cells, and representing hemopoietic, endothelial, muscle and connective tissue phenotypes. The procoagulant activity on each cell type was characterised by the use of specifically immunodepleted plasmas and specific inhibitors, including monoclonal antibodies. The predominant cell surface trigger of coagulation found in this series was tissue factor, and only blood monocytes provided some evidence for direct activation of factor X independent of FVII. Human ECV304 transformed endothelial cells were more closely studied as representative of a cell type constitutively expressing procoagulant. Coagulation mediated by ECV304 cells was found to be strictly dependent on tissue factor, as shown by an inhibitory monoclonal antibody, and on coagulation factors V, VII and X. ECV304 procoagulant activity was strongly inhibited by active-site-inactivated FVIIa, a synthetic peptide inhibitor of FXa (Tenstop) and the thrombin inhibitor, hirudin. While not appropriate for routine clinical assessment of coagulation factor function, we have found this model to be valuable in characterising the procoagulant activity on different cell types and particularly useful as a drug discovery tool in the search for new anticoagulants.

Effects of ionic and nonionic contrast media on endothelium and on arterial thrombus formation.

BACKGROUND: The aims of the present study were to investigate whether ionic and nonionic contrast media (CM) affect: 1) the procoagulant and fibrinolytic activities of cultured human vessel endothelium; and 2) early events of tissue-factor-induced arterial thrombus formation under conditions which may follow a percutaneous transluminal coronary angioplasty (PTCA) procedure. The following 3 CM were studied: iohexol (nonionic monomer, Omnipaque); iodixanol (nonionic dimer, Visipaque); and ioxaglate (ionic dimer, Hexabrix). Saline (0.9%) and glucose (40 vol%) were used as control. METHODS AND RESULTS: Exposing endothelium to 40 vol% CM for 10 min did not affect the selected parameters of cellular procoagulant (tissue factor), anticoagulant (thrombomodulin), fibrinolytic (tissue plasminogen activator) or antifibrinolytic (plasminogen activator inhibitor-1) activity or antigen. However, ioxaglate had a profound impact on the cell morphology, which was noted already after one minute of exposure. The cells contracted and rounded, exposing large areas of extracellular matrix. Iohexol showed this phenomenon to a considerably lesser extent, whereas iodixanol induced a slight swelling of the cells without detectable exposure of extracellular matrix. The effect of the respective CM on tissue-factor-driven thrombus formation at an arterial shear rate of 2600 s-1 was studied in an ex vivo parallel-plate perfusion chamber device. In this model, human native blood was passed over a tissue factor/phospholipid-rich surface following 30 s exposure to 100% CM. The CM was washed out by nonanticoagulated blood drawn directly from an antecubital vein by a pump positioned distal to the perfusion chamber. Such a pre-exposure of the procoagulant surface to iodixanol reduced the fibrin deposition around the platelet thrombi by 50% (p<0.01). However, iohexol and ioxaglate did not affect fibrin deposition. None of the 3 CM affected the recruitment of platelets in the thrombi, since similar values were obtained with pre-exposure to 40 vol% of saline. CONCLUSION: Iodixanol appears to be most biocompatible with endothelium, and has a moderate inhibitory effect on fibrin deposition in flowing blood. This differs from iohexol, and in particular from ioxaglate, which induce endothelial changes in morphology with no effect on fibrin deposition. Since none of the CM affected the platelet aggregate formation, and since ioxaglate has been reported to have stronger anticoagulant and antithrombotic properties than iodixanol or iohexol in in vitro assays, it is apparent that these properties were not reflected in thrombus formation under the experimental conditions of high arterial shear.

Induction of monocyte tissue factor procoagulant activity during coronary artery bypass surgery is reduced with heparin-coated extracorporeal circuit.

The possible activation of monocytes to express tissue factor procoagulant activity (TF-PCA) during CPB (cardiopulmonary bypass) was investigated. 22 patients undergoing myocardial revascularization were randomly assigned to two groups. In group C, heparin-coated circuits (Duraflo II) and reduced systemic heparinization (ACT > 250s) were used. In group NC, non-coated circuits and standard heparin administration (ACT > 480s) were used. Adherent monocytes retrieved from the oxygenators immediately after bypass arrest showed a 2-3-fold increase in TF-PCA when compared to circulating cells pre-CPB (P < 0.01). When cell PCA was expressed as percent change from pre-CPB (baseline) values, circulating monocytes in group NC at CPB-arrest showed a 2-fold increase in PCA compared to group C (P < 0.05). Moreover, the percent increase in PCA of oxygenator-retrieved monocytes was 7-fold in group NC and 2-fold in group C (P < 0.008 and P < 0.004, respectively). Thus, heparin-coating of the extracorporeal circuit reduced induction of adherent cell TF-PCA by 70% (P < 0.05). Thus, monocyte TF-PCA may cause activation of the extrinsic coagulation pathway during CPB surgery. It is apparent that heparin-coating enhanced biocompatibility of extracorporeal circuits. Reduced systemic heparinization in group C proved to be safe. However, further reduction of heparin administration may not be advisable, since monocytes were still activated in the coated oxygenator.

Reduced effect of aspirin on thrombus formation at high shear and disturbed laminar blood flow.

Aspirin is the most commonly used antithrombotic drug in primary and secondary prophylaxis against cardio- and cerebrovascular disease. In previous studies from our laboratory it was demonstrated that the effect of aspirin on collagen-induced thrombus formation in a parallel- plate perfusion device with laminar blood flow is shear rate dependent. Although aspirin did not affect collagen-induced thrombus formation at 650 s-1 (medium sized arteries), a significant inhibition of thrombus formation by approximately 38% at 2,600 s-1 (moderately stenoses in medium sized arteries) was observed. At present we have extended these studies to thrombus formation at the apex of eccentric stenoses in a parallel-plate perfusion chamber device. The stenoses reduced the cross-sectional area of the blood flow channel of the perfusion chambers by 60 or 80%, introducing disturbed laminar flow and apex wall shear rates of 2,600 and 10,500 s-1, respectively. The corresponding wall shear stresses were 80 and 315 dynes/cm2, respectively. Aspirin reduced the platelet thrombus volume at the 60% stenosis by 45% (p < 0.03), and the fibrin deposition by 70% (p < 0.004). However, none of these parameters were affected by aspirin at the 80% stenosis. These observations may at least partly explain why aspirin has a limited clinical effect in preventing arterial thrombus formation in atherosclerotic vessels at high shear and disturbed blood flow. In contrast, thrombus formation in blood from one patient with Glanzmann's thrombasthenia and two patients with von Willebrand disease subtype 2M was almost abolished at this blood flow condition. Thus, blocking the function of either von Willebrand factor or glycoprotein IIb/IIIa may represent better antithrombotic approaches for such critical events than blocking the prostaglandin metabolism by aspirin. The lack of effect of aspirin on thrombus formation at the 80% stenosis may reflect shear-induced platelet activation at the stenosis inlet region, since shear-induced platelet aggregation in rotational viscometers is not affected by aspirin at shear stresses exceeding 100 dynes/cm2.

Retinoic acid reduces induction of monocyte tissue factor and tissue factor/factor VIIa-dependent arterial thrombus formation.

Agents that downregulate the induction of monocyte/macrophage tissue factor (TF) activity may attenuate the thrombotic risk associated with mechanical restoration of vessel patency or artificial arterial grafting. In such events, procoagulant macrophages in the atherosclerotic plaque and procoagulant monocytes adherent to artificial materials may be exposed to the blood stream. Ishii et al (Blood 80:2556, 1992) reported that induction of endothelial TF is downregulated by all-trans retinoic acid (ATRA), and Conese et al (Thromb Haemost 66:662, 1991) reported that retinoids downregulate monocyte procoagulant activity (PCA). These findings led us to investigate the effect of ATRA on monocyte TF expression, and to study the effect of ATRA on monocyte-induced thrombus formation in a model system of human arterial thrombogenesis. Induction of PCA in human peripheral blood monocytes by 0.5 microgram/mL lipopolysaccharide (LPS) was dose dependently reduced by ATRA, reaching a reduction of 56% at 10(-5) mol/L ATRA (P < .0001). A 38% reduction (P < .0007) in LPS-induced TF antigen expression was observed at an ATRA concentration of 10(-6) mol/L. Adherence of monocytes to plastic cover slips (Thermanox, Miles Laboratories, Naperville, IL) also triggered induction of cellular PCA, which was inhibited by more than 80% by an anti-TF monoclonal antibody (MoAb) (P < .002). Inclusion of ATRA (10(-6) mol/L) reduced this PCA by 40% (P < .03), and the TF antigen expression by 30% (P < .0001). Exposure of Thermanox adherent monocytes to flowing nonanticoagulated human blood in a parallel-plate perfusion chamber device at an arterial wall shear rate of 650 s-1 elicited significant fibrin deposition and platelet thrombus formation. Partial interruption of this thrombus formation was achieved by 10(-6) mol/L ATRA, which reduced the fibrin deposition by 80% (P < .02) and platelet thrombus formation by 50% (P < .05). In comparison, incubation of adherent monocytes with the anti-TF MoAb before the blood exposure, reduced the fibrin deposition by 83% (P < .02) and platelet thrombus volume by 75% (P < .0008). Thus, ATRA is an effective down-regulator of monocyte TF-PCA, and may reduce thrombotic complications at sites of plaque rupture, at plaque disruption after percutaneous transluminal angioplasty procedures, or on surfaces introduced by artificial arterial grafting.

Procoagulant human monocytes mediate tissue factor/factor VIIa-dependent platelet-thrombus formation when exposed to flowing nonanticoagulated human blood.

Tissue factor (TF) on monocyte and macrophage surfaces is a nonproteolytic cofactor for factor VIIa (FVIIa)-induced coagulation. Monocyte-derived macrophages in atherosclerotic plaques express TF, which, after plaque disruption or rupture, may complex with FVII/VIIa from the bloodstream, resulting in activation of extrinsic coagulation. We studied the effect of TF expression on human monocytes on arterial thrombus formation in a model system of thrombogenesis. Thawed, cryopreserved human monocytes adherent to plastic coverslips were stimulated with lipopolysaccharide (0.5 microgram/mL) to express TF and subsequently exposed to flowing nonanticoagulated human blood in a parallel-plate perfusion chamber. The wall shear rate at the cell surface was 650 seconds-1, corresponding to that of average-sized coronary arteries. The stimulated monocytes elicited pronounced fibrin deposition and platelet-thrombus formation. The platelet-thrombus volume was as large as that triggered by human type III collagen fibrils under similar experimental conditions. In contrast, the monocytes elicited much more fibrin deposition than the collagen surface. However, inclusion of an anti-TF monoclonal antibody that blocks the complexation of FVII/FVIIa with TF virtually abolished the fibrin deposition (P < .03) and reduced platelet-thrombus formation by more than 70% (P < .04). Thus, arterial thrombus formation induced by stimulated monocytes was almost completely blocked by the anti-TF antibody, suggesting that inhibition of TF/FVIIa complex formation on monocytes and macrophages at sites of plaque rupture or after percutaneous transluminal coronary angioplasty procedures may reduce intravascular thrombotic complications.