Parallel-plate Perfusion Chamber Research Articles

Reduced Thrombus Formation in Native Blood of Homozygous Factor VII-Deficient Patients at High Arterial Wall Shear Rate

Inhibition of thrombin formation in flowing native blood reduces thrombus formation on subendothelium, dacron, or collagen fibrils at arterial wall shear rates of 450 to 650 s-1. In the present study, we have investigated the role of low levels of factor VII (FVII) in thrombus formation on collagen fibrils at arterial wall shear rates of 650 s-1 (coronary arteries), 2,600 s-1 (mildly stenosed arteries), and 10,510 s-1 (severely stenosed arteries) in parallel-plate perfusion chambers. In the perfusion chamber with the highest wall shear rate, thrombus formation took place at the apex of an eccentric stenosis, which reduced the cross-sectional area of the blood flow channel by 80%, thus simulating thrombus formation at an atherosclerotic plaque rupture. Native blood from 21 healthy volunteers and 12 homozygous FVII-deficient patients was drawn by a pump directly from an antecubital vein over a surface of fibrillar collagen positioned in the respective perfusion chambers. The patients had FVII coagulant activities ranging from 1.3% to 4.5% and FVII antigen levels of 16% to 23% of normal. Immunoaffinity purification of the patients' FVII followed by electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]) and immunoblotting showed a protein with similar molecular mass as normal FVII. In the perfusion studies, a reduction in thrombus volume of 54% of normal (P < .007) at 10,510 s-1 was observed. The deposition of fibrin on the thrombogenic surface and the plasma level of fibrinopeptide A (FPA) in blood samples collected distal to the perfusion chamber were concomitantly reduced (P < .002 and P < .04, respectively). The plasma FPA level was also reduced at 2,600 s-1 (P < .04), but not at 650 s-1. However, at the lower shear conditions, the thrombus volume and the fibrin deposition were within the ranges observed in normal blood. The platelet-collagen adhesion was not affected at any of the three shear conditions. Thus, low plasma levels of FVII result in significantly less formation of thrombin and fibrin in and around growing platelet masses at high shear condition. This may weaken the thrombus stability and reduce platelet recruitment, thereby lowering thrombus volume. In support of this theory, one patient with afibrinogenemia had an 83% reduction in thrombus volume at this high shear condition.

Reduced thrombus formation in native blood of homozygous factor VII- deficient patients at high arterial wall shear rate

Inhibition of thrombin formation in flowing native blood reduces thrombus formation on subendothelium, dacron, or collagen fibrils at arterial wall shear rates of 450 to 650 s-1. In the present study, we have investigated the role of low levels of factor VII (FVII) in thrombus formation on collagen fibrils at arterial wall shear rates of 650 s-1 (coronary arteries), 2,600 s-1 (mildly stenosed arteries), and 10,510 s-1 (severely stenosed arteries) in parallel-plate perfusion chambers. In the perfusion chamber with the highest wall shear rate, thrombus formation took place at the apex of an eccentric stenosis, which reduced the cross-sectional area of the blood flow channel by 80%, thus simulating thrombus formation at an atherosclerotic plaque rupture. Native blood from 21 healthy volunteers and 12 homozygous FVII- deficient patients was drawn by a pump directly from an antecubital vein over a surface of fibrillar collagen positioned in the respective perfusion chambers. The patients had FVII coagulant activities ranging from 1.3% to 4.5% and FVII antigen levels of 16% to 23% of normal. Immunoaffinity purification of the patients' FVII followed by electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]) and immunoblotting showed a protein with similar molecular mass as normal FVII. In the perfusion studies, a reduction in thrombus volume of 54% of normal (P &lt; .007) at 10,510 s-1 was observed. The deposition of fibrin on the thrombogenic surface and the plasma level of fibrinopeptide A (FPA) in blood samples collected distal to the perfusion chamber were concomitantly reduced (P &lt; .002 and P &lt; .04, respectively). The plasma FPA level was also reduced at 2,600 s-1 (P &lt; .04), but not at 650 s-1. However, at the lower shear conditions, the thrombus volume and the fibrin deposition were within the ranges observed in normal blood. The platelet-collagen adhesion was not affected at any of the three shear conditions. Thus, low plasma levels of FVII result in significantly less formation of thrombin and fibrin in and around growing platelet masses at high shear condition. This may weaken the thrombus stability and reduce platelet recruitment, thereby lowering thrombus volume. In support of this theory, one patient with afibrinogenemia had an 83% reduction in thrombus volume at this high shear condition.

Effect of Blood Flow on Thrombin Generation Is Dependent on the Nature of the Thrombogenic Surface

Abstract 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 &lt; .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 &lt; .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.

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.

Modulation of thrombotic responses in moderately stenosed arteries by cigarette smoking and aspirin ingestion.

Cigarette smoking is a known risk factor for cardiovascular disease in men and women, and it has been suggested that this risk is linked to enhanced formation of platelet thromboxane A2 (TxA2). This led us to investigate the effect of cigarette smoking and TxA2 formation on collagen-induced thrombogenesis in flowing nonanticoagulated human blood. Thrombus formation in blood from smokers and nonsmokers was compared before and 2 hours after ingestion of a single oral dose of 990 mg aspirin, which is sufficient to block platelet TxA2 formation. Nonanticogulated blood was drawn directly from an antecubital vein over collagen fibrils in a parallel-plate perfusion chamber by a peristaltic roller pump placed distally to the chamber. Wall shear rates at the collagen surface were characteristic for medium-sized (650 s-1) and moderately stenosed (2600 s-1) arteries. Blood-collagen interactions were morphologically quantified, and markers of platelet release, beta-thromboglobulin (beta-TG), and activation of coagulation, fibrinopeptide A (FPA), were measured immediately distal to the perfusion chamber. The thrombus volume in blood from cigarette-smoking individuals was nearly twofold larger than in blood from nonsmokers at 2600 s-1 (37.4 and 19.4 microns 3/microns 2; P < .03). However, ingestion of aspirin reduced the thrombus volume in blood from smokers by 61.8% (P < .01), which was substantially more than the 37.6% reduction in blood from nonsmokers (P < .03). Neither cigarette smoking nor aspirin ingestion affected thrombus formation at 650 s-1. The plasma levels of FPA and beta-TG were slightly lower in nonsmokers and after aspirin ingestion.(ABSTRACT TRUNCATED AT 250 WORDS)

Clopidogrel – a Platelet Inhibitor which Inhibits Thrombogenesis in Non-Anticoagulated Human Blood Independently of the Blood Flow Conditions

The goal of the present study was to investigate the effect of 7 and 14 days of daily oral administration of 75 mg clopidogrel on collagen-induced thrombogenesis in flowing non-anticoagulated human blood. Blood was drawn directly from an antecubital vein over immobilised collagen type III fibrils positioned in a parallel-plate perfusion chamber. The wall shear rates at the collagen surface were those characteristic for veins (100 s-1), and for medium sized (650 s-1) and moderately stenosed (2600 s-1) arteries. Clopidogrel ingestion reduced the thrombus volume significantly (p < 0.05) at 100 and 2600 s-1 (39 and 51% respectively). The beta-thromboglobulin plasma levels were reduced concomitantly. However, it was not possible to measure accurately the thrombus volume at 650 s-1, due to loose packing of the platelet thrombi. Transmission electron microscopy substantiated this observation and showed that clopidogrel profoundly reduced the platelet degranulation process (p < 0.005). The inhibitory effect of clopidogrel on platelet consumption by the growing thrombi resulted apparently in higher platelet concentration at the collagen surface, which enhanced the platelet-collagen adhesion at all three shear rates (p < 0.05). Despite the low deposition of fibrin on collagen, clopidogrel reduced significantly the fibrinopeptide A plasma levels and the fibrin deposition at shear rates below 650 s-1. This was apparently a consequence of the reduced platelet recruitment and the lower activation of platelets, since activated platelets in thrombi promote deposition of fibrin.(ABSTRACT TRUNCATED AT 250 WORDS)

HN-11500 – a Novel Thromboxane A2 Receptor Antagonist With Antithrombotic Activity in Humans at Arterial Blood Flow Conditions

In the present study we have investigated the effect of a 100 mg single oral dose of a newly developed thromboxane A2 receptor antagonist on collagen-induced thrombogenesis in flowing human non-anticoagulated blood. Blood was drawn directly from an antecubital vein over immobilised collagen type III fibrils on a cover slip placed in a parallel-plate perfusion chamber. Shear rates at the collagen surface were characteristic for medium sized (650 s-1) and moderately stenosed (2,600 s-1) arteries. Blood-collagen interactions were morphologically quantified as platelet-collagen adhesion, fibrin deposition and thrombus volume. Activation peptides of coagulation, fibrinopeptide A (FPA), and of platelets, beta-thromboglobulin (beta-TG), were measured immediately distal to the perfusion chamber. HN-11500 ingestion reduced significantly the thrombus volume by 32% at 2,600 s-1, but not at 650 s-1. However, transmission electron microscopy revealed loosely packed and less degranulated platelets at 650 s-1. The beta-TG plasma levels were also reduced at both shear rates by the HN-11500 ingestion. The platelet-collagen adhesion was significantly enhanced at both shear rates. This was apparently a consequence of higher platelet concentrations at the collagen surface, because fewer platelets were consumed by the thrombi after the drug ingestion. In contrast, the coagulation, as measured by fibrin deposition and FPA plasma levels, was not significantly affected by HN-11500. Thus, it appears that the thromboxane A2 receptor antagonist HN-11500 reduces the thrombotic response by primarily impairing the platelet function at arterial blood flow conditions, and particularly at high wall shear rates.

Modulation of procoagulant activity of extracellular endothelial matrix by anti-tissue factor antibody and the synthetic peptide Arg-Gly-Asp-Val. Experiments with flowing nonanticoagulated human blood

Fibrin forms on and binds to the extracellular matrix of endotoxin-stimulated endothelium when exposed to flowing non-anticoagulated blood. These processes have been investigated by employing a human ex vivo perfusion model, a synthetic peptide Arg-Gly-Asp-Val and a monoclonal anti-tissue factor antibody which inhibits tissue factor/FVIIa-induced coagulation. Procoagulant extracellular matrix on plastic cover-slips was prepared from cultures of endotoxin-stimulated human endothelium following brief exposure to 0.1 M NH4OH. Non-anticoagulated blood was drawn directly from an antecubital vein by a pump at venous (100/s) and arterial (650/s) wall shear rates over the matrix-coated cover-slips positioned in parallel-plate perfusion chambers. Deposition of fibrin and platelets on the matrix was quantified by morphometry. Preincubation of the matrix with Arg-Gly-Asp-Val inhibited fibrin deposition by 80-90% at both venous (P < 0.001) and arterial shear (P < 0.05). However, the peptide had no effect on the clotting time in a modified one-stage clotting assay where coagulation was initiated by lysed endotoxin-stimulated endothelial cells, indicating that the peptide interfered with the binding of fibrin to the matrix in the perfusion model. Preincubation of the matrix with the anti-tissue factor antibody, which blocked the coagulant activity ( > 95%, P < 0.01) in the modified coagulation assay, also inhibited fibrin deposition on the matrix by 90-95% (P < 0.01) at both shear rates. In the absence of either inhibitor, platelets adhered preferentially to the fibrin meshwork, and more so at arterial shear. Platelet thrombus formation on the fibrin coat was in particular pronounced at arterial shear. Thus, it appears that the extracellular matrix of endotoxin-stimulated endothelium initiates coagulation predominantly through tissue factor/FVIIa and that the resulting fibrin meshwork forming on the surface induces rapid platelet thrombus formation. The inhibitory effect of Arg-Gly-Asp-Val on the binding of fibrin to the matrix may indicate the presence of specific matrix fibrinogen/fibrin binding site(s) with a recognition sequence of Arg-Gly-Asp.

Human type II hyperlipoproteinemia enhances platelet-collagen adhesion in flowing nonanticoagulated blood.

We investigated the effects of high plasma lipid levels on platelet adhesion and platelet thrombus formation in nonanticoagulated human blood on collagen fibrils at an arterial wall shear rate of 2600 seconds-1. Nonanticoagulated blood was drawn directly at a flow rate of 10 mL/min for 3 minutes from an antecubital vein of patients with type IIa (n = 5) and type IIb (n = 4) hyperlipoproteinemia over purified human type III collagen fibrils that were positioned on a plastic coverslip in a parallel-plate perfusion chamber. Results were compared with those obtained in healthy individuals with normal lipid plasma levels (n = 9). Blood-collagen interactions were quantified by morphometry as platelet-collagen adhesion, thrombus volume, and fibrin deposition. Platelet-collagen adhesion in the two groups of patients was significantly higher than in healthy individuals (70.7 [61.2 to 82.0] and 70.3 [66.4 to 81.0] in types IIa and IIb patients, respectively, versus 51.2 [44.5 to 68.6] in control subjects; P < .05. All values are percent median [range]). In contrast, the thrombus volume was similar in the three groups (11.3 [8.0 to 13.0], 9.6 [6.4 to 15.3], and 10.2 [6.8 to 16.1] microns3/microns2 [range], respectively). Differences in fibrin deposition were not observed. Thus, it appears that platelet-collagen adhesion is augmented in patients with type IIa and IIb hyperlipoproteinemia, indicating that the process of thrombogenesis is hastened in these patients.

Aggregate formation is more strongly inhibited at high shear rates by dRGDW, a synthetic RGD-containing peptide.

The effect of D-Arg-Gly-Asp-Trp (dRGDW), a synthetic RGD-containing peptide, on platelet adhesion and aggregate formation on various purified adhesive proteins and the extracellular matrix of endothelial cells was investigated with anticoagulated blood recirculating through a parallel-plate perfusion chamber. Aggregate formation on the extracellular matrix of phorbol myristate acetate (PMA)-stimulated endothelial cells and on collagen type I was more strongly inhibited by dRGDW at higher shear rates than at a low shear rate. Platelet adhesion to the extracellular matrix of nonactivated and PMA-stimulated endothelial cells was inhibited by dRGDW, especially at high shear rates, probably as a consequence of the inhibition of platelet spreading. Inhibition by dRGDW of platelet adhesion to von Willebrand factor, fibronectin, and fibrinogen was almost complete, indicating that platelet adhesion to these substrates is mediated through RGD-directed receptors. Platelet adhesion to laminin was not inhibited by the peptide, whereas platelet adhesion to collagen was increased as a consequence of the inhibition of aggregate formation. Our results show that dRGDW is a strong inhibitor of platelet adhesion and aggregate formation, especially at high shear rates.

Platelets lacking functional CD36 (glycoprotein IV) show reduced adhesion to collagen in flowing whole blood

A parallel-plate perfusion chamber has been used to evaluate the contribution of the adhesive membrane glycoprotein CD36 (GPIV) to platelet adhesion on type I collagen in flowing whole blood at a shear rate of 800 s-1. In one series of experiments, reconstituted normal blood (hematocrit 0.4; platelet count 1.5 x 10(5)/microL) was prepared from washed red blood cells, plasma, and washed platelets that had been incubated with Fab fragments of a monospecific polyclonal anti-CD36 antibody (50 micrograms/mL, 30 minutes, 37 degrees C). Percent surface coverage of collagen-coated coverslips using reconstituted blood with antibody-blocked platelets, as compared with paired reconstituted controls (100%), was 50% at 2 minutes, 87% at 5 minutes, and 90% at 10 minutes. Further studies were performed by perfusion of whole blood from a healthy donor of the Naka-negative phenotype, whose platelets constitutively lack CD36, over collagen-coated coverslips. In this case, percent surface coverage was 55% of normal controls at 2 minutes, 76% of controls at 5 minutes, and 72% of controls at 10 minutes. In both preparations, platelets lacking functional CD36 had a statistically significant decrease (P &lt; .005) in adhesion after 2 minutes and 10 minutes perfusion but not at 5 minutes. These results show that functional CD36 facilitates the rapid adhesion of platelets to collagen and that this effect is seen at the earliest time points of their interaction.

Platelets Lacking Functional CD36 (Glycoprotein IV) Show Reduced Adhesion to Collagen in Flowing Whole Blood

AbstractA parallel-plate perfusion chamber has been used to evaluate the contribution of the adhesive membrane glycoprotein CD36 (GPIV) to platelet adhesion on type I collagen in flowing whole blood at a shear rate of 800 s–1. In one series of experiments, reconstituted normal blood (hematocrit 0.4; platelet count 1.5 × 105/μL) was prepared from washed red blood cells, plasma, and washed platelets that had been incubated with Fab fragments of a monospecific polyclonal anti-CD36 antibody (50 µg/mL, 30 minutes, 37°C). Percent surface coverage of collagen-coated coverslips using reconstituted blood with antibody-blocked platelets, as compared with paired reconstituted controls (100%), was 50% at 2 minutes, 87% at 5 minutes, and 90% at 10 minutes. Further studies were performed by perfusion of whole blood from a healthy donor of the Naka-negative phenotype, whose platelets constitutively lack CD36, over collagen-coated coverslips. In this case, percent surface coverage was 55% of normal controls at 2 minutes, 76% of controls at 5 minutes, and 72% of controls at 10 minutes. In both preparations, platelets lacking functional CD36 had a statistically significant decrease (P < .005) in adhesion after 2 minutes and 10 minutes perfusion but not at 5 minutes. These results show that functional CD36 facilitates the rapid adhesion of platelets to collagen and that this effect is seen at the earliest time points of their interaction.

Inhibition of platelet adhesion and thrombus formation on a collagen-coated surface by novel carbamoylpiperidine antiplatelet agents

The inhibitory effect of two novel antiplatelet agents, 1,10-bis-( N,N-diethylcarbamoyl)piperidono]decane dihydrobromide (G-110) and 1,6bis[3-( N,N-diethylcarbamoyl)piperidino]hexane dihydrobromide (G-112) on platelet adhesion and subsequent aggregation on collagen-coated surface was evaluated under controlled flow. Glass coverslips coated with bovine fibrillar collagen type I were exposed to heparinized human whole blood that had been preincubated with either aqueous solutions of one of the two carbamoylpiperidine congeners or corresponding amounts (1.0–4.0 μl/ml blood) of distilled water, at a wall shear rate of 1000 s −1, in a parallel-plate perfusion chamber. Epifluorescence video microscopy with a microphotometric measurement technique was used to visualize and quantify deposition of fluorescently-labeled platelets from flowing whole blood onto the collagen-coated surface. At concentrations of 100 and 200 μM, G-110 inhibited platelet accumulation by 30±9% (±S.E.) and 63±3% (±SE), respectively; while G-112 reduced platelet deposition by 19±3% (±SE) and 31±2% (±SE) at concentrations of 200 and 400 μM, respectively. Digital image processing techniques were used to analyze the dynamics of thrombus growth on the collagen-coated surfaces. It was found that the compounds reduced the rate of thrombus growth by impeding both surface coverage and the number of platelets per thrombus in a concentration-dependent manner. This study, together with others on related compounds GT-12, BPAT-117 and BPAT-143, corroborates the nature of pivotal features in the molecular structure of carbamoylpiperidine and nipecotoylpiperazine derivatives which enhance desirable antithrombotic effects, e.g. intramolecular distance between two tertiary amines (ring nitrogens) and levels of hydrophobicity.

Shear rate-dependent impairment of thrombus growth on collagen in nonanticoagulated blood from patients with von Willebrand disease and hemophilia A

Thrombus formation on collagen fibrils was quantified at venous (100/s) and arterial (650/s and 2,600/s) wall shear rates in blood from patients with various subtypes of von Willebrand disease (vWD) and with hemophilia A (HA). Nonanticoagulated blood was drawn directly from an antecubital vein over purified type III collagen fibrils exposed in parallel-plate perfusion chambers. Blood-collagen interactions were differentiated and quantified by morphometry as platelet adhesion, thrombus height, thrombus volume, and deposition of fibrin strands. Sixteen patients with vWD, including four type III, six type I, four type IIA, and two type IIB, were compared with 26 normal subjects and nine patients with HA, including six severe HA and three mild HA. Platelet adhesion and thrombus formation at 2,600/s were significantly decreased in blood from patients with vWD type III, IIA, and IIB, but not in blood from patients with type I and in HA. The abnormal thrombus formation was apparently not related to the decreased levels of factor VIII (F.VIII), because thrombus height and volume were normal in severe and mild HA. Thrombus formation at 650/s was also significantly decreased in patients with vWD type III, IIA, and IIB and slightly reduced in type I. Significant reduction in thrombus volume and height was also observed in blood from patients with severe HA, but not in mild HA. Thrombus dimensions were not affected at 100/s in the vWD subtypes. However, significantly decreased thrombus height and virtually absent fibrin deposition were observed in blood from patients with severe HA. Apparently, F.VIII supports thrombus formation at low and intermediate shear conditions, presumably through the generation of thrombin. In contrast, von Willebrand factor (vWF) mediates not only platelet adhesion, but also thrombus formation at intermediate and high shear rates. Thus, the relative contribution of coagulation (F.VIII) and platelet function (vWF) in thrombus formation appears to be shear rate dependent, but having optimal effects at different shear conditions.

Shear Rate-Dependent Impairment of Thrombus Growth on Collagen in Nonanticoagulated Blood From Patients With von Willebrand Disease and Hemophilia A

AbstractThrombus formation on collagen fibrils was quantified at venous (100/s) and arterial (650/s and 2,600/s) wall shear rates in blood from patients with various subtypes of von Willebrand disease (vWD) and with hemophilia A (HA). Nonanticoagulated blood was drawn directly from an antecubital vein over purified type III collagen fibrils exposed in parallel-plate perfusion chambers. Blood-collagen interactions were differentiated and quantified by morphometry as platelet adhesion, thrombus height, thrombus volume, and deposition of fibrin strands. Sixteen patients with vWD, including four type III, six type I, four type IIA, and two type IIB, were compared with 26 normal subjects and nine patients with HA, including six severe HA and three mild HA. Platelet adhesion and thrombus formation at 2,600/s were significantly decreased in blood from patients with vWD type III, IIA, and IIB, but not in blood from patients with type I and in HA. The abnormal thrombus formation was apparently not related to the decreased levels of factor VIII (F.VIII), because thrombus height and volume were normal in severe and mild HA. Thrombus formation at 650/s was also significantly decreased in patients with vWD type III, IIA, and IIB and slightly reduced in type I. Significant reduction in thrombus volume and height was also observed in blood from patients with severe HA, but not in mild HA. Thrombus dimensions were not affected at 100/s in the vWD subtypes. However, significantly decreased thrombus height and virtually absent fibrin deposition were observed in blood from patients with severe HA. Apparently, F.VIII supports thrombus formation at low and intermediate shear conditions, presumably through the generation of thrombin. In contrast, von Willebrand factor (vWF) mediates not only platelet adhesion, but also thrombus formation at intermediate and high shear rates. Thus, the relative contribution of coagulation (F.VIII) and platelet function (vWF) in thrombus formation appears to be shear rate dependent, but having optimal effects at different shear conditions.

Aurin Tricarboxylic Acid Inhibits Platelet Adhesion to Collagen by Binding to the 509-695 Disulphide Loop of von Willebrand Factor and Competing with Glycoprotein Ib

Aurin tricarboxylic acid (ATA) is known to inhibit ristocetin-induced platelet agglutination but not arachidonic acid-, epinephrine- or ADP-induced aggregation. Its capacity to abolish human von Willebrand factor (vWF)-platelet interactions was further investigated by measurement of platelet adhesion to collagen, platelet agglutination tests and binding studies. In flowing blood using parallel-plate perfusion chambers and human collagen, ATA inhibited platelet adhesion to completion in a dose-dependent manner only at the highest shear rate tested (2,600 s-1). It was without effect at 100 and 650 s-1. ATA completely abolished vWF-dependent platelet agglutination induced by ristocetin, botrocetin and asialo-vWF, respectively. 125I-vWF binding to ristocetin- and botrocetin-treated platelets, to heparin and to sulfatides as well as 125I-botrocetin binding to vWF was competitively inhibited by ATA. By contrast, binding of 125I-vWF to collagen was not affected. To further localize the domain of vWF interacting with ATA, experiments of inhibition of binding of selected 125I-monoclonal antibodies (MoAbs) to immobilized vWF by ATA were performed. Our data led to the conclusion that: 1) the interaction of ATA with vWF involves sequences of the A1 disulphide loop of vWF (residues 509-695) and close epitopes which interact with GPIb and 2) the inhibition of platelet adhesion by ATA occurs only at a high shear rate where vWF is known to play a key role. Thus ATA, which blocks the vWF/GPIb pathway by interfering with vWF and not with platelets, is a potential tool in preventing the early stages of thrombosis.

Role of von Willebrand Factor Associated to Extracellular Matrices in Platelet Adhesion

The respective role of plasmatic and endothelial extracellular matrix (ECM)-associated von Willebrand factor (vWF) in platelet adhesion was investigated at a high shear rate using a parallel-plate perfusion chamber. Incubation of the endothelial ECM with a monoclonal antibody (MoAb) to vWF, which specifically blocks vWF binding to platelet GP Ib (MoAb 322), inhibited 45% of platelet adhesion. Complete inhibition was achieved by incubating both plasma and endothelial ECM with MoAb 322 at concentrations that blocked only about 50% of adhesion when added separately. The effect of ECM-associated vWF was further demonstrated when a fibroblastic ECM, normally devoid of vWF, was coated with purified plasmatic vWF. Matrix associated-vWF was able to significantly enhance platelet adhesion in both the presence and the absence of plasmatic vWF. In contrast, this effect was not seen on endothelial ECM. Binding of exogenous vWF to the ECM was specific and dose dependent, reached the same value (500 ng/cm2) on both fibroblastic ECM and endothelial ECM, but exhibited a threefold-lower apparent dissociation constant (KD) on fibroblastic than on endothelial ECM. Our studies suggest that vWF deposited by endothelial cells in the ECM may be the most active form in platelet adhesion, whereas plasmatic vWF may only play a secondary role.

Role of von Willebrand factor associated to extracellular matrices in platelet adhesion

The respective role of plasmatic and endothelial extracellular matrix (ECM)-associated von Willebrand factor (vWF) in platelet adhesion was investigated at a high shear rate using a parallel-plate perfusion chamber. Incubation of the endothelial ECM with a monoclonal antibody (MoAb) to vWF, which specifically blocks vWF binding to platelet GP Ib (MoAb 322), inhibited 45% of platelet adhesion. Complete inhibition was achieved by incubating both plasma and endothelial ECM with MoAb 322 at concentrations that blocked only about 50% of adhesion when added separately. The effect of ECM-associated vWF was further demonstrated when a fibroblastic ECM, normally devoid of vWF, was coated with purified plasmatic vWF. Matrix associated-vWF was able to significantly enhance platelet adhesion in both the presence and the absence of plasmatic vWF. In contrast, this effect was not seen on endothelial ECM. Binding of exogenous vWF to the ECM was specific and dose dependent, reached the same value (500 ng/cm2) on both fibroblastic ECM and endothelial ECM, but exhibited a threefold-lower apparent dissociation constant (KD) on fibroblastic than on endothelial ECM. Our studies suggest that vWF deposited by endothelial cells in the ECM may be the most active form in platelet adhesion, whereas plasmatic vWF may only play a secondary role.

Baboon Fibrinogen Adsorption and Platelet Adhesion to Polymeric Materials

The role of fibrinogen in mediating platelet adhesion to polymers exposed to blood plasma was studied by comparison of the effect of plasma dilution on fibrinogen adsorption and platelet adhesion, and by the use of coagulation factor deficient plasmas. Polyetherurethane substrates were first preadsorbed with dilute plasma, then contacted with washed platelets suspended in a modified, apyrase containing Tyrode's buffer. Platelet adhesion was studied under static conditions in Multiwell dishes, and also under shearing conditions using a parallel plate perfusion chamber. Fibrinogen adsorption and platelet adhesion were measured using 125I radiolabeled baboon fibrinogen and 111In radiolabeled baboon platelets, respectively. Surfaces were characterized by electron spectroscopy for chemical analysis (ESCA). When fibrinogen adsorption to Biomer was measured after 2 h contact with a series of dilute plasma solutions under static conditions, a peak in adsorption was observed from 0.26% plasma, i.e., adsorption was greater from 0.26% plasma than from either more or less dilute plasma. A peak in subsequent platelet adhesion to the plasma preadsorbed surfaces, measured after 2 h static incubation with washed platelets, was also observed but occurred on Biomer preadsorbed with 1.0% plasma. When fibrinogen adsorption was measured after 5 min contact under shearing conditions, the fibrinogen adsorption peak occurred on surfaces that had been exposed to 1.0% plasma. A peak in platelet adhesion to these preadsorbed surfaces, measured after 5 min contact with the platelet suspensions under shearing conditions, was observed on Biomer preadsorbed with 0.1% plasma. Shifts between the positions of the peaks in protein adsorption and platelet adhesion occurred on other polymers tested as well. Platelet adhesion was almost completely inhibited when baboon and human plasmas lacking fibrinogen (i.e., serum, heat defibrinogenated plasma, and congenitally afibrinogenemic plasma) were used. Platelet adhesion was restored to near normal when exogenous fibrinogen was added to fibrinogen deficient plasmas. Adhesion was also inhibited completely when a monoclonal antibody directed against the glycoprotein IIb/IIIa complex was added to the platelet suspension. Platelet adhesion to surfaces preadsorbed to von Willebrand factor deficient plasma was the same as to surfaces preadsorbed with normal plasma. While it appears that surface bound fibrinogen does mediate the initial attachment of platelets to Biomer, the observation that the fibrinogen adsorption and platelet adhesion maxima do not coincide exactly also suggests that the degree of subsequent platelet adhesion is dictated not only by the amount of surface bound fibrinogen but also by its conformation.

Collagen type III induced ex vivo thrombogenesis in humans. Role of platelets and leukocytes in deposition of fibrin.

Exposure of type III collagen coats on plastic cover slips in parallel-plate perfusion chambers to flowing nonanticoagulated human blood resulted in deposition of platelets and fibrin. Blood was drawn directly from an antecubital vein by an occlusive roller pump over the collagen coats in chambers having flow slits of different dimensions, so that wall shear rates of 100, 650, and 2600 s-1 were obtained at 10 ml/min. Coagulation was minimally activated during the passage of blood from the vein to the chamber as shown by fibrinopeptide A levels of 3.7 ng/ml after 5-minute perfusions. The surface coverage with platelets increased from 18% at 100 s-1 to 59% at 2600 s-1, and the corresponding thrombus volumes increased from 2 to 22 microns 3/microns 2, respectively. This contrasted with the coverage with fibrin on collagen, which decreased from 28% at 100 s-1 to 9% at 2600 s-1. Fibrin deposits on the thrombi covered 6% of the surface irrespective of the shear rate, indicating that some of the deposited platelets accelerated the deposition of fibrin. The type III collagen preparation did not activate factor XII and did not possess tissue factor activity, indicating that the surface itself was not procoagulant. However, a correlation between deposited leukocytes and surface coverage with fibrin was observed (r = 0.78, p less than 0.01), suggesting a role for these cells in the deposition of fibrin. The data demonstrate that thrombogenesis is triggered by pure type III collagen, although the deposition of fibrin is not initiated by the collagen itself but presumably by deposited leukocytes.(ABSTRACT TRUNCATED AT 250 WORDS)