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.

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Tick anticoagulant peptide (TAP) is a potent and selective inhibitor of factor Xa. TAP has shown good antithrombotic efficacy in experimental animal models of disseminated intravascular coagulation and venous and arterial thrombogenesis. In the present study we evaluated the effect of recombinant TAP (rTAP) on acute thrombus formation in human nonanticoagulated blood triggered either by tissue factor (TF) or by collagen at arterial shear conditions. The main goal was to establish the role of factor Xa in thrombus formation by use of an optimal inhibitory concentration of rTAP. Blood was drawn directly from an antecubital vein by a pump over the respective thrombogenic surfaces, which were positioned in a parallel-plate perfusion chamber. rTAP was mixed homogeneously into the flowing blood by a heparin-coated device positioned proximal to the perfusion chamber. The passage of blood through this device caused minor activation of coagulation but little activation of platelets. Fibrinopeptide A and beta-thromboglobulin levels after 5 minutes of blood perfusion were, on average, 14 ng/mL and 45 IU/mL, respectively. rTAP at a plasma concentration of 0.90 mumol/L completely inhibited TF/factor VIIa-dependent thrombus formation at wall shear rates of 650 and 2600 s-1. These shear conditions are comparable to those in medium-sized arteries and in moderately stenosed small arteries, respectively. In contrast to the TF-coated surface, rTAP was less efficient in reducing collagen-induced thrombus formation. While a significant reduction of 53% was observed at 650 s-1, thrombus formation at 2600 s-1 was not affected by rTAP.(ABSTRACT TRUNCATED AT 250 WORDS)

The relative importance of vessel wall tissue factor (TF) in initiating thrombogenesis is not well defined. In contrast , vessel wall collagens have been well documented as potent inducers of thrombus formation. We compared the potency of a human TF/phospholipid surface with that of a surface consisting of human type III collagen fibrils in triggering thrombus formation in native human blood at venous and arterial blood flow conditions. A commercial preparation, Thromborel S, was used as a source of human TF. Biochemical characterization of this preparation revealed small amounts of FVII, FIX, and FX proteins. Coagulant activity of these proteins was associated with the FVII protein only, although it was a very low activity. Studies with anti-TF antibodies in a one-stage clotting assay showed that the procoagulant activity of Thromborel was mainly a result of TF. The molar ratio of TF to phospholipid was 1:2 x 10 7. Thrombus formation in flowing nonanticoagulated human blood drawn directly from an antecubital vein was triggered by either Thromborel S or collagen fibrils coated on Thermanox coverslips in a parallel-plate perfusion chamber device. A 1:50 Thromborel S dilution gave maximal fibrin T issue factor (TF) initiates coagulation through its interaction with the plasma coagulation protein FVTI/FVIIa. 12 Activated FVII (FVIIa) bound to TF activates the coagulation proteins FX and FIX, 34 which ultimately results in thrombin generation, fibrin formation, and platelet activation and aggregation. The wall shear rate has a profound impact on TF/ FVIIa-dependent FX activation, 5-7 as well as on fibrin deposition and platelet thrombus formation. 815 The significance of TF for fibrin deposition and thrombus formation on subendothelium, 7-13 ' 16 stimulated endothe-lial cells, 1718 and the extracellular matrix of stimulated endothelium 15 has been investigated in nonanticoagu-lated human blood by various perfusion chamber devices. These experiments have revealed a significant decrease in fibrin deposition with increasing shear and a concomitant enhancement of platelet thrombus formation. Immobilized purified TF reconstituted in phospho-lipid vesicles and TF expressed on fibroblasts have also been exposed to purified FVII and FX in flowing deposition (90% surface coverage) at a wall shear rate of 100 s"'. However, pretreatment of the TF surface with a monoclonal anti-TF antibody reduced this fibrin deposition by 93% (P<.001). Thus, TF was essential for the procoagulant activity of the Thromborel S surface in this flow system also. At higher wall shear rates (650 and 2600 s" 1), less fibrin was deposited, but the platelet thrombus formation on the fibrin mesh increased dramatically. Platelet thrombus formation was elicited on top of the fibrin mesh; platelets adhered exclusively to the fibrin strands formed on the Thromborel S surface. However, while much less fibrin was deposited on the collagen (/ > <.001), the platelet-collagen adhesion was substantially higher than the platelet-fibrin adhesion on the Thromborel S ...

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 A 2 (TxA 2 ). This led us to investigate the effect of cigarette smoking and TxA 2 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 TxA 2 formation. Nonanticoagulated blood was drawn directly from an antecubital vein over collagen fibrils in a parallel-plate perfusion chamber by a peristaltic roller pump placed distalry 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, £-thromboglobulin (0-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 S everal prospective studies have demonstrated that cigarette smoking is a major risk factor for cardiovascular disease. 16 Enhanced thromboxane A 2 (TxA 2 ) synthesis in platelets has been implicated as a possible source of risk, since biochemical studies have shown increased TxA 2 production in cigarette smokers. 7The TxA 2 molecule is a potent vasoconstrictor and platelet aggregation agonist. Both events are closely associated with cardiovascular disorders.A single oral dose of 1 g aspirin blocks the platelet production of TxA 2 8 by irreversible acetylation of platelet cyclooxygenase. 9 The effect of blocking TxA 2 is partial inhibition of the aggregation response to low concentrations of ADP and collagen. Ingestion of aspirin abolishes acute and chronic effects of enhanced platelet aggregation observed in healthy habitual smokers. -11The goal of the present study was to investigate the effect of cigarette smoking on collagen-induced thrombogenesis in flowing nonanticoagulated human blood and to evaluate the effect of a 990-mg single oral dose of aspirin in both smokers and nonsmokers at arterial blood flow conditions. A well-characterized human ex