Impact of fibronectin assembly on platelet thrombus formation in response to type I collagen and von Willebrand factor

Plasma fibronectin enhances platelet thrombus formation on surfaces coated with collagen. We investigated the role of fibronectin assembly in this process. Platelets adherent to fibrillar type I collagen, but not platelets adherent to von Willebrand factor (VWF), supported assembly of plasma fibronectin under static conditions. At a shear rate of 1250 s(-1), platelets adherent to collagen assembled coperfused plasma fibronectin and formed larger thrombi in a fibronectin-concentration-dependent manner, with a maximum effect at 250 mug/mL. Enhanced thrombus formation on collagen was blocked by a peptide that binds to the N-terminal region of fibronectin and inhibits fibronectin assembly. Cross-linking of fibronectin to collagen prior to exposure to platelets had no effect on thrombus formation. Collagen-induced platelet thrombus formation at a shear rate of 5000 s(-1) required coperfusion with VWF and did not result in assembly of coperfused fibronectin. VWF-mediated increase in platelet thrombi on collagen was not enhanced and indeed was somewhat attenuated by coperfused fibronectin at a shear rate of 5000 s(-1). These results indicate that, at moderately high but not very high shear rates, fibronectin assembly in platelet aggregates that form in response to collagen enhances thrombus formation and serves as an alternative to VWF-mediated enhancement.     

Dissociation between fibrinogen and fibrin interaction with platelets in patients with different subtypes of Glanzmann's thrombasthenia: studies in an ex vivo perfusion chamber model

To explore the possible role of a residual or variant alphaIIbbeta3 integrin (alphaIIbbeta3) in thrombogenesis, we used a new ex vivo perfusion chamber model to examine blood from patients with different subtypes of Glanzmann's thrombasthenia (GT). Non-anticoagulated blood was perfused through capillaries coated with type III collagen for 4.5 min (shear rate: 1600/s). Platelet deposition was quantified as platelet adhesion and mean thrombus size volume; fibrin and von Willebrand Factor (VWF) were specifically revealed by immunohistochemistry. In two patients with variant and in one patient with type II GT, platelet adhesion was maximal and we observed an unexpected formation of thrombi that were smaller than normal in size. These thrombi were surrounded by a thick meshwork that displayed a strong staining for fibrin and VWF. In two patients with heterozygous GT, platelet adhesion and thrombogenesis were normal. In two patients with type I GT, there was no thrombus formation, although platelet adhesion was also maximal. These data suggest the existence of a substitute pathway for thrombogenesis mediated by fibrin and possibly alphaIIbbeta3 (alphaIIbbeta3 at a reduced level, as in type II, and/or abnormal) as this fibrin network was not observed in type I GT with no alphaIIbbeta3. These interactions might facilitate haemostasis and even lead to thrombosis under certain favourable conditions. Furthermore, these data might have pharmacological relevance to the development of anti-alphaIIbbeta3 antithrombotic agents.     

Effect of blood flow on thrombin generation is dependent on the nature of the thrombogenic surface

We have investigated the influence of blood flow on thrombin generation, fibrin formation, and fibrin deposition on procoagulant and nonprocoagulant surfaces. Nonanticoagulated human blood was drawn for 5 minutes directly from an antecubital vein over stimulated endothelial cells expressing tissue factor and over human type III collagen fibrils, positioned in parallel-plate perfusion chambers. The shear rates at these surfaces were 50, 650, and 2,600 s-1. Deposition of platelets and fibrin was measured by morphometry. Thrombin and fibrin formation was determined by measuring prothrombin fragments 1 + 2 (F 1 + 2), thrombin-antithrombin III complexes, (T-AT) and fibrinopeptide A (FPA) in blood effluent from the perfusion chamber at the end of the 5- minute perfusion period. On procoagulant endothelial cells, the thrombi were primarily composed of fibrin. The fibrin deposition (81%, 21%, and 2% at 50, 650, and 2,600 s-1, respectively) and plasma levels of F 1 + 2, T-AT and FPA were shear rate dependent and highest at 50 s-1. There was a positive correlation between F 1 + 2 and T-AT and the fibrin deposition (P < .01). In contrast, the collagen surface triggered primarily thrombi that were composed of platelets. The platelet thrombi and plasma levels of F 1 + 2 and T-AT were also dependent on the shear rate, but highest at 650 and 2,600 s-1. F 1 + 2 and T-AT reached the same level as observed with procoagulant endothelial cells at the higher shear rates. There was a positive correlation between F 1 + 2 and T-AT and the platelet thrombus formation (P < .05), confirming the predominant role of platelets in thrombin generation. Thus, thrombin formation is strongly influenced by the blood flow, and this effect depends on the composition of the thrombogenic surface.     

Effects of aspirin and prostaglandin E1 on in vitro thrombolysis with urokinase. Evidence for a possible role of inhibiting platelet activity in thrombolysis

The formation of thrombi in vivo includes the activation of both platelets and the coagulation cascade. Conventional thrombolytic therapy is primarily directed toward the dissolution of fibrin. To evaluate the possibility that platelet activity impairs the lysis of thrombi, we studied the effects of aspirin and platelet-deaggregating prostaglandin E1 on thrombolysis with urokinase. Combined platelet and fibrin thrombi were produced in vitro by adding CaCl2 and collagen (1 microgram/ml) to citrated platelet-rich plasma (250,000 platelets per microliters). Urokinase (500-10,000 units/ml) caused a dose-dependent weight loss of the thrombi that was maximal at 2,000 units/ml. The addition of aspirin (10-200 micrograms/ml) to platelet-rich plasma before thrombus formation markedly enhanced thrombolysis with urokinase. This effect was most pronounced at 20 micrograms/ml aspirin. However, when aspirin was added after completion of thrombus formation, no significant effect on thrombolysis was noted. Prostaglandin E1 (1-100 mumol/l) improved the lysis with urokinase of the combined platelet and fibrin thrombi. This effect was maximal at 20 mumol/l prostaglandin E1. When pure fibrin thrombi were produced in platelet-free plasma, prostaglandin E1 was without effect on lysis. Thus, in vitro lysis with urokinase of combined platelet and fibrin thrombi was enhanced by the addition of platelet-deaggregating prostaglandin E1 and by pretreatment with aspirin.     

Fibronectin is required for platelet adhesion and for thrombus formation on subendothelium and collagen surfaces

Fibronectin (FN) plays a role in several adhesion mediated functions including the interaction of platelets with subendothelium. We investigated the role of plasma FN in platelet adhesion and platelet thrombus formation under flow conditions. We used two different perfusion models: the annular chamber with alpha-chymotrypsin-treated rabbit vessel segments, and the flat chamber with coverslips coated with fibrillar purified human collagen type III. Perfusates consisted of washed platelets and washed RBCs, suspended in normal or FN-depleted plasma. Perfusions were carried out for ten minutes at shear rates of 300 or 1,300 s-1. Platelet deposition and thrombus dimensions were evaluated morphometrically by a computerized system. We found that depletion of plasma fibronectin significantly reduced the percentage of total coverage surface and percentage of platelet thrombus, at both shear rates studied, and in both perfusion systems (P less than .01) (P less than .01). The dimensions of the platelet thrombi formed in perfusions at high shear rate were also significantly reduced in perfusions carried out with FN depleted plasma (P less than .01). Addition of purified FN to FN-depleted perfusates restored all values to those measured in the control perfusions. These results indicate that plasma FN is required for platelet aggregate and thrombus formation following adhesion under flow conditions.     

Two distinct mechanisms are involved in stent thrombosis under flow conditions

The mechanism of stent thrombosis was evaluated by a flow chamber model. A Palmaz-Schatz stent was placed on glass surfaces coated with type I collagen in a parallel-plate flow chamber. Platelets were rendered fluorescent with mepacrine and whole blood was perfused through the chamber. Platelet thrombi formed around the implanted stents were continuously recorded by epi-fluorescent video-microscopy. Platelet thrombi appeared on the collagen surface within a few minutes in the absence of function blocking monoclonal antibodies. Antibody blocking vWF interaction with GP Ib f completely inhibited the platelet thrombi around the stent, but had no effect on platelet thrombus formation in the space between the stents and collagen, while both were inhibited by anti-GP IIb/IIIa. Our results suggest that two distinct mechanisms, one mediated by vWF interaction with GP Ib f occurring at high shear rates, and the other mediated exclusively by GP IIb/IIIa occurring at low shear rates, are involved in the process of platelet thrombus formation in the presence of stents.     

Control of thrombus embolization and fibronectin internalization by integrin alpha IIb beta 3 engagement of the fibrinogen gamma chain

Fibrin(ogen) deficiency (Fg-/-) was shown previously to be compatible with rapid thrombus growth within injured arterioles, but platelet fibronectin content was increased and newly formed thrombi were unstable. To further define the role of fibrin(ogen) in thrombus formation and stabilization, platelet biology was examined in mice expressing a form of fibrinogen that clots normally but lacks the gamma chain C-terminal binding site for alpha IIb beta 3 (Fg gamma Delta 5). Thrombus growth within the arterioles of Fg gamma Delta 5 mice appeared faster than in wild-type mice despite a far greater emboli formation. Unlike Fg-/- mice, the emboli were relatively small and released from the top of thrombi, rather than by fracture at the vessel wall. The fibronectin content in Fg gamma Delta 5 platelets was also dramatically increased through a beta 3 integrin-dependent mechanism. The following has been concluded: (1) Fibrin formation contributes to, but is not sufficient for, the stabilization of arterial thrombi. Platelet receptor engagement of the C-terminal of the Fg gamma chain contributes to the stable incorporation of platelets into thrombi. (2) Alternative ligands to fibrinogen can support efficient thrombus growth. (3) Fibrinogen is internalized through alpha IIb beta 3 engagement of the fibrinogen gamma chain element, and this interaction secondarily controls the fibronectin content of platelets.     

Comparison of the antithrombotic effect of PEG-hirudin and heparin in a human ex vivo model of arterial thrombosis.

Polyethylene glycol (PEG)-hirudin is a derivative of hirudin with a long plasma half-life. We have compared the efficacy of PEG-hirudin with unfractionated heparin (UH) in preventing arterial thrombosis. Arterial thrombus formation was induced ex vivo in 12 healthy human volunteers by exposing a tissue factor-coated coverslip positioned in a parallel-plate perfusion chamber to flowing nonanticoagulated human blood drawn directly from an antecubital vein at an arterial wall shear rate of 2600 s-1 for 3.5 minutes. PEG-hirudin, UH, or saline (as control) were administered ex vivo through a heparin-coated mixing device positioned proximal to the perfusion chamber. Platelet and fibrin deposition was quantified by immunoenzymatic measure of the P-selectin and D-dimer content of dissolved plasmin-digested thrombi, respectively. UH was administered to a plasma concentration of 0.35 IU/mL. This concentration prolonged the activated partial thromboplastin time from 32+/-1 seconds to 79+/-4 seconds (P<0.01). UH did not significantly prevent platelet deposition. However, fibrin deposition was reduced by 39% (P<0.05). PEG-hirudin in plasma concentrations of 0.5, 2.5, and 5 microg/mL prolonged the activated partial thromboplastin time to 48+/-2, 87+/-4, and 118+/-4 seconds, respectively. In contrast to UH, PEG-hirudin prevented both platelet and fibrin deposition in a dose-dependent manner with a >80% reduction at 5 microg/mL (P<0.01). Furthermore, the plasma level of PEG-hirudin required to significantly prevent fibrin deposition (0.5 microg/mL) corresponded to a much shorter prolongation of activated partial thromboplastin time (48+/-2 seconds) than that needed for UH (79+/-4 seconds). Thus, our results are compatible with the view that thrombin is greatly involved in recruitment of platelets in evolving thrombi, and that PEG-hirudin is an effective agent for preventing arterial thrombosis in a human ex vivo experimental model.     

Rate-limiting roles of the tenase complex of factors VIII and IX in platelet procoagulant activity and formation of platelet-fibrin thrombi under flow

The importance of factor Xa generation in thrombus formation has not been studied extensively so far. Here, we used mice deficient in either factor VIII or factor IX to determine the role of platelet-stimulated tenase activity in the formation of platelet-fibrin thrombi on collagen. With tissue factor present, deficiency in factor VIII or IX markedly suppressed thrombus growth, fibrin formation and platelet procoagulant activity (phosphatidylserine exposure). In either case, residual fibrin formation was eliminated in the absence of tissue factor. Effects of factor deficiencies were antagonized by supplementation of the missing coagulation factor. In wild-type thrombi generated under flow, phosphatidylserine-exposing platelets bound (activated) factor IX and factor X, whereas factor VIII preferentially co-localized at sites of von Willebrand factor binding. Furthermore, proteolytic activity of the generated activated factor X and thrombin was confined to the sites of phosphatidylserine exposure. With blood from a hemophilia A or B patient, the formation of platelet-fibrin thrombi was greatly delayed and reduced, even in the presence of high concentrations of tissue factor. A direct activated factor X inhibitor, rivaroxaban, added to human blood, suppressed both thrombin and fibrin formation. Together, these data point to a potent enforcement loop in thrombus formation due to factor X activation, subsequent thrombin and fibrin generation, causing activated factor X-mediated stimulation of platelet phosphatidylserine exposure. This implies that the factor VIII/factor IX-dependent stimulation of platelet procoagulant activity is a limiting factor for fibrin formation under flow conditions, even at high tissue factor concentrations.     

The impact of factor Xa inhibition on axial dependent arterial thrombus formation triggered by a tissue factor rich surface

This study was designed to assess the effect of Factor Xa antagonists on thrombus formation at various axial positions on a tissue factor rich surface under arterial blood flow conditions. Non-anticoagulated, flowing human blood, drawn directly from an antecubital vein, was perfused over a tissue factor coated cover slip in a parallel-plate perfusion chamber. Thrombus surface coverage, thrombus mean height and fibrin surface coverage were measured at six different axial positions by confocal microscopy. Both thrombus surface coverage and mean height decreased along the cover slip axis whereas the fibrin surface coverage increased. Pre-chamber treatment of blood with the direct Factor Xa inhibitors Razaxaban and 813893 resulted in significantly reduced thrombus and fibrin formation at all axial positions investigated (P < 0.05). Thrombus and fibrin deposition in a laminar flow chamber changed with axial position with surface coverage measurements being more reproducible than thrombus mean height. Data were more reproducible towards the centre of the flow chamber than at the extremities. Razaxaban and 813893 inhibited thrombus and fibrin formation at the highest concentrations tested. No difference in drug effect was apparent at different axial positions. In conclusion, axial position influences the degree of thrombus and fibrin deposition with measurements being less reproducible at the extremities of the flow chamber. This technique may prove useful for analysing anti-thrombotic drug effects before progression to clinical trials.     

Kinetics of urokinase-induced thrombolysis in a biphasic in vitro system

The role and effect of added lys-plasminogen (lys-PLG) on urokinase-induced thrombolysis in an in vitro biphasic system were investigated. The kinetics of lysis of whole blood thrombi was followed in perfusion mediums of normal plasma, PLG-deficient plasma and normal saline using a high and a low concentration of urokinase (UK). The lysis of standard whole blood thrombi in whole blood perfusion mediums to which had been added UK alone or UK plus lys-PLG was compared to whole blood thrombi enriched with lys-PLG by incorporation during thrombus formation or by adsorption during perfusion. In addition, the kinetics of lysis of PLG-deficient fibrin thrombi perfused in PLG-deficient plasma or normal saline was studied when lys-PLG had been added to the thrombus, to the perfusion medium or to both thrombus and medium. In PLG-deficient plasma from which plasmin inhibitors had not been removed, thrombolysis was minimal even at a high concentration of UK. This effect could be neutralized, and to some extent, regulated, by lys-PLG enrichment of the medium. Both PLG-incorporated and PLG-adsorbed whole blood thrombi gave initial and sustained acceleration of UK-induced lysis in comparison with standard nontreated thrombi. It is concluded that in a blood-thrombus biphasic thrombolytic system induced by UK, there is interaction between the phases, and that PLG in both phases influences thrombolysis.     

Par4 is required for platelet thrombus propagation but not fibrin generation in a mouse model of thrombosis

Thrombin, a central mediator of hemostasis and thrombosis, converts fibrinogen to fibrin and is a potent platelet activator. Activated platelets provide a surface for assembly of the tenase and prothrombinase complexes required for thrombin generation. The role of thrombin-induced platelet activation in platelet accumulation and its interplay with fibrin deposition during thrombus assembly has not been fully defined. We studied these processes during laser-induced thrombus formation by using real-time digital fluorescence microscopy in mice lacking protease-activated receptor-4 (Par4), which is necessary for thrombin responsiveness in mouse platelets. Juxtamural platelet accumulation immediately after laser injury was not different in wild-type and Par4(-/-) mice. However, subsequent growth of platelet thrombi was markedly diminished in Par4(-/-) mice. At the time of maximal thrombus size in wild type, platelet accumulation was more than 10-fold higher in wild type than in Par4(-/-) mice. P-selectin expression, a marker of platelet activation, was reduced and delayed in Par4(-/-) thrombi. In contrast to platelet activation and accumulation, the rate and amount of fibrin deposition, predominantly intramural and juxtamural in this model, were indistinguishable in Par4(-/-) and wild-type mice. These results suggest that platelet activation by thrombin is necessary for normal propagation of a platelet thrombus at a distance from the injured vessel wall and hence for normal thrombus growth. However, platelet activation by thrombin is unnecessary for initial and limited accumulation of platelets at or near the vessel wall, and this limited accumulation of platelets and/or platelet-independent mechanism(s) of thrombin generation are sufficient for normal fibrin deposition in this model.     

Role of collagen-adherent platelets in mediating fibrin formation in flowing whole blood

Activated platelets provide assembly sites for coagulation enzyme complexes and in this way can mediate coagulation during hemostasis and thrombosis. In this study, we examined the procoagulant activity of platelets adhering directly to fibrillar collagen, a main thrombogenic constituent of subendothelium. For this purpose, we used a human ex- vivo thrombosis model in which collagen-coated coverslips were exposed to flowing nonanticoagulated blood (shear rate, 65/s) for 5.5 minutes, which led to the deposition of adherent platelets, platelet thrombi, and fibrin. To examine the procoagulant activity of adherent platelets only, a selective antagonist of the platelet GPIIb-IIIa complex, Ro 44- 9883, was infused via a mixing device, resulting in a complete abrogation of platelet thrombus formation but leaving the collagen- adherent platelet layer intact. This platelet layer generated increased postchamber fibrinopeptide A (FPA) levels (203 +/- 33 ng/mL) as compared with control experiments without infusion of inhibitor (95 +/- 13 ng/mL). Concomitantly, fibrin deposition measured by morphometric analysis of cross-sections was also increased, as was the platelet adhesion to collagen. An immunochemical staining of fibrin fibers further showed that the adherent platelets formed the nuclei for fibrin fiber formation. This increase in fibrin deposition was mediated by the intrinsic factor X (F.X) activation complex on adherent single platelets, because almost complete inhibition of FPA generation (9 ng/mL) and fibrin deposition (0.4% +/- 0.2% coverage) was achieved upon coinfusion of the GP IIb-IIIa antagonist and active site-inhibited F.IXa. The large platelet thrombi that were deposited in control experiments contained no significant amounts of immunodetectable fibrin except at the thrombus base, where adherent platelets anchored the thrombi to the collagen surface. These results suggest that the collagen-adherent platelets are important promoters of coagulation during the initial phase of thrombogenesis by providing assembly sites for the F.X activation complex.     

Extracellular DNA traps promote thrombosis

Neutrophil extracellular traps (NETs) are part of the innate immune response to infections. NETs are a meshwork of DNA fibers comprising histones and antimicrobial proteins. Microbes are immobilized in NETs and encounter a locally high and lethal concentration of effector proteins. Recent studies show that NETs are formed inside the vasculature in infections and noninfectious diseases. Here we report that NETs provide a heretofore unrecognized scaffold and stimulus for thrombus formation. NETs perfused with blood caused platelet adhesion, activation, and aggregation. DNase or the anticoagulant heparin dismantled the NET scaffold and prevented thrombus formation. Stimulation of platelets with purified histones was sufficient for aggregation. NETs recruited red blood cells, promoted fibrin deposition, and induced a red thrombus, such as that found in veins. Markers of extracellular DNA traps were detected in a thrombus and plasma of baboons subjected to deep vein thrombosis, an example of inflammation-enhanced thrombosis. Our observations indicate that NETs are a previously unrecognized link between inflammation and thrombosis and may further explain the epidemiological association of infection with thrombosis.     

Blood Fibrinolytic Activity during Arvin Therapy

When Arvin is administered to patients, there is a rapid fall in plasma plasminogen concentration and fibrin degradation products appear in the blood which can be detected both by their anticoagulant effect and by their reaction with anti-fibrinogen serum. There is a minimal but inconstant increase in the concentration of plasminogen activator and circulating plasmin cannot be detected. These findings are similar to those recorded in the defibrination syndrome and are explained by the assumption that both plasminogen and activator are adsorbed onto intravascular fibrin micro-clots. Local plasmin release would then cause lysis of fibrin with the appearance of fibrin degradation products.
There is no apparent reason why this mechanism should assist in the lysis of coexisting preformed thrombi. The apparent rapid lysis of thrombi which has been observed in some patients treated with Arvin can be explained if such thrombi are not static, but represent a balance between fibrin deposition and fibrinolysis. Therapeutic defibrination, by preventing extension of the thrombus, would allow the normal fibrinolytic mechanism to produce dissolution of the thrombus.     

Hematopoietic cell-derived microparticle tissue factor contributes to fibrin formation during thrombus propagation

Tissue factor (TF) is expressed on nonvascular cells and cells within the vessel wall and circulates in blood associated with microparticles. Although blood-borne TF accumulates into the developing thrombus during thrombus formation, the contribution of blood-borne TF and vessel wall TF to thrombin generation in vivo following vessel injury is unknown. To determine the source and role of blood-borne microparticle TF, we studied arterial thrombus formation in a living mouse using intravital microscopy. Platelet, TF, and fibrin accumulation in the developing thrombus was compared in wild-type and low TF mice. Compared to wild-type mice, low TF mice formed very small platelet thrombi lacking TF or fibrin. Wild-type and low TF mice received transplants of bone marrow from wild-type and low TF mice. Arterial thrombi in low TF bone marrow/wild-type chimeric mice had decreased size and decreased TF and fibrin levels. Arterial thrombi in wild-type bone marrow/low TF chimeric mice showed decreased platelet thrombus size but normal TF and fibrin levels. This demonstrates that blood-borne TF associated with hematopoietic cell-derived microparticles contributes to thrombus propagation.     

Prevention of vascular graft occlusion and thrombus-associated thrombin generation by inhibition of factor XI

The protease thrombin is required for normal hemostasis and pathologic thrombogenesis. Since the mechanism of coagulation factor XI (FXI)-dependent thrombus growth remains unclear, we investigated the contribution of FXI to thrombus formation in a primate thrombosis model. Pretreatment of baboons with a novel anti-human FXI monoclonal antibody (aXIMab; 2 mg/kg) inhibited plasma FXI by at least 99% for 10 days, and suppressed thrombin-antithrombin (TAT) complex and beta-thromboglobulin (betaTG) formation measured immediately downstream from thrombi forming within collagen-coated vascular grafts. FXI inhibition with aXIMab limited platelet and fibrin deposition in 4-mm diameter grafts without an apparent increase in D-dimer release from thrombi, and prevented the occlusion of 2-mm diameter grafts without affecting template bleeding times. In comparison, pretreatment with aspirin (32 mg/kg) prolonged bleeding times but failed to prevent graft occlusion, supporting the concept that FXI blockade may offer therapeutic advantages over other antithrombotic agents in terms of bleeding complications. In whole blood, aXIMab prevented fibrin formation in a collagen-coated flow chamber, independent of factor XII and factor VII. These data suggest that endogenous FXI contributes to arterial thrombus propagation through a striking amplification of thrombin generation at the thrombus luminal surface.     

Plasma fibronectin promotes thrombus growth and stability in injured arterioles

Mice lacking both of the best-known platelet ligands, von Willebrand factor and fibrinogen, can still form occlusive thrombi in injured arterioles. The platelets of these animals accumulate excessive amounts of fibronectin (FN). These observations led us to examine the contribution of plasma FN (pFN) to thrombus formation. Inactivation of the FN gene in FN conditional knockout mice reduced pFN levels to <2% and platelet FN to approximately 20% of the levels in similarly treated control mice. The mice were then observed in a model of arterial injury to evaluate their capacity to form thrombi. The deficiency of pFN did not affect the initial platelet adhesion, but a delay of several minutes in thrombus formation was observed in the arterioles of pFN-deficient mice as compared with control mice. The thrombi that formed in the absence of pFN were stably anchored to the vessel wall but continuously shed platelets or small platelet clumps, thus slowing their growth significantly; the platelet/platelet cohesion was apparently diminished. Consequently the occlusion of pFN-deficient vessels was delayed, with the majority of vessels remaining patent at the end of the 40-min observation period. We conclude that, in addition to von Willebrand factor and fibrinogen, FN plays a significant role in thrombus initiation, growth, and stability at arterial shear rates and that deficiency in each of the three platelet ligands has its own specific impact on platelet plug formation.     

ADAMTS-13 attenuates thrombus formation on type I collagen surface and disrupted plaques under flow conditions

Plaque disruption with subsequent thrombus formation is a major cause of atherothrombotic diseases and von Willebrand factor (VWF), which is cleaved by ADAMTS-13, plays a critical role in thrombus formation. However, the role of ADAMTS-13 during thrombogenesis on atherosclerotic vessel remains unknown. We examined the localization of ADAMTS-13 in coronary thrombi obtained from patients with acute myocardial infarction. We also investigated the roles of ADAMTS-13 in thrombus formation using type I collagen-coated flow chambers (100S(-1) and 1500S(-1)) and on injured neointima of rabbit femoral arteries. ADAMTS-13 was present in thrombi of human coronary arteries, where it co-localized with VWF. In a flow chamber, both the average of the surface covered by platelet adhesion and the long axes of platelet thrombi were significantly augmented by an antibody to the ADAMTS-13 disintegrin-like domain (WH2-22-1A) at a shear rate of 1500s(-1), but not by an antibody to the ADAMTS-13 thrombospondin 1-3 domain (WH10). WH2-22-1A also reduced the activity of plasma ADAMTS-13 to cleave large VWF multimers during perfusion. Thrombi on injured neointima were induced by repeated balloon injury of rabbit femoral arteries, and were composed of platelet and fibrin, like human coronary thrombi. WH2-22-1A significantly augmented thrombus formation on injured neointima. These results suggest that the disintegrin-like domain of ADAMTS-13 functions in attenuating thrombus growth on diseased arteries exposed to a high shear rate.     

Verotoxin-1-induced up-regulation of adhesive molecules renders microvascular endothelial cells thrombogenic at high shear stress

Verotoxin-1 (VT-1)-producing Escherichia coli is the causative agent of postdiarrheal hemolytic uremic syndrome (D+HUS) of children, which leads to renal and other organ microvascular thrombosis. Why thrombi form only on arterioles and capillaries is not known. This study investigated whether VT-1 directly affected endothelial antithrombogenic properties promoting platelet deposition and thrombus formation on human microvascular endothelial cell line (HMEC-1) under high shear stress. Human umbilical vein endothelial cells (HUVECs) were used for comparison as a large-vessel endothelium. HMEC-1 and HUVECs were pre-exposed for 24 hours to increasing concentrations of VT-1 (2-50 pM) and then perfused at 60 dynes/cm(2) with heparinized human blood prelabeled with mepacrine. Results showed that VT-1 significantly increased platelet adhesion and thrombus formation on HMEC-1 in comparison with unstimulated control cells. An increase in thrombus formation was also observed on HUVECs exposed to VT-1, but to a remarkably lower extent. The greater sensitivity of HMEC-1 to the toxin in comparison with HUVECs was at least in part due to a higher expression of VT-1 receptor (20-fold more) as documented by FACS analysis. The HMEC-1 line had a comparable susceptibility to the thrombogenic effect of VT-1 as primary human microvascular cells of the same dermal origin (HDMECs). The adhesive molecules involved in VT-induced thrombus formation were also studied. Blocking the binding of von Willebrand factor to platelet glycoprotein Ib by aurintricarboxylic acid (ATA) or inhibition of platelet alpha(IIb)beta(3)-integrin by chimeric 7E3 Fab resulted in a significant reduction of VT-1-induced thrombus formation, suggesting the involvement of von Willebrand factor-platelet interaction at high shear stress in this phenomenon. Functional blockade of endothelial beta(3)-integrin subunit, vitronectin receptor, P-selectin, and PECAM-1 with specific antibodies was associated with a significant decrease of the endothelial area covered by thrombi. Confocal microscopy studies revealed that VT-1 increased the expression of vitronectin receptor and P-selectin and redistributed PECAM-1 away from the cell-cell border of HMEC-1, as well as of HDMECs, thus indicating that the above endothelial adhesion molecules are directly involved and possibly determine the effect of VT-1 on enhancing platelet adhesion and thrombus formation in microvascular endothelium. These results might help to explain why thrombi in HUS localize in microvessels rather than in larger ones and provide insights on the molecular events involved in the process of microvascular thrombosis associated with D+HUS.