Non-anticoagulated Blood Research Articles

A Double-Blind Randomized Comparison of Combined Aspirin and Ticlopidine Therapy Versus Aspirin or Ticlopidine Alone on Experimental Arterial Thrombogenesis in Humans

No randomized study comparing the effect of combined ticlopidine and aspirin therapy versus each drug alone in reducing poststenting thrombotic complications has been performed. To compare these three antiplatelet regimens versus placebo, we conducted a double-blind randomized study using an ex vivo model of thrombosis. Sixteen healthy male volunteers were assigned to receive for 8 days the following four regimens separated by a 1-month period: aspirin 325 mg/d, ticlopidine 500 mg/d, aspirin 325 mg/d + ticlopidine 500 mg/d, and placebo. At the end of each treatment period, native nonanticoagulated blood was drawn directly from an antecubital vein over collagen- or tissue factor (TF)-coated coverslips positioned in a parallel-plate perfusion chamber at an arterial wall shear rate (2,600 s−1 ) for 3 minutes. Thrombus, which formed on collagen in volunteers treated by placebo, were rich in platelets and poor in fibrin. As compared with placebo, aspirin and ticlopidine alone reduced platelet thrombus formation by only 29% and 15%, respectively (P > .2). In contrast, platelet thrombus formation was blocked by more than 90% in volunteers treated by aspirin + ticlopidine (P < .01v placebo or each treatment alone). Furthermore, the effect of the drug combination therapy was significantly larger than the sum of the two active treatments (P < .05). Thrombus, which formed on TF-coated coverslips in volunteers treated by placebo, were rich in fibrin and platelets. Neither of the three antiplatelet treatments significantly inhibited fibrin deposition and platelet thrombus formation on this surface (P > .2). Thus, the present study shows that combined aspirin and ticlopidine therapy dramatically potentiates the antithrombotic effect of each drug alone, but that the antithrombotic effect of the combined treatment depends on the nature of the thrombogenic surface.© 1998 by The American Society of Hematology.

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A New Method for Quantifying Platelet Deposition in Flowing Native Blood in an Ex Vivo Model of Human Thrombogenesis

No quantitative, simple and non-radioactive method has been described for measuring the platelet content of experimental thrombi. The aim of the present study was to develop a simple method for quantifying platelets in thrombi formed on thrombogenic surfaces in flowing native human blood. To test the relevance of this new method, the effect of unfractionated heparin on arterial thrombus formation was investigated. Tissue factor (TF)- and collagen-coated coverslips were exposed to non-anticoagulated blood at an arterial wall shear rate (2,600 s(-1)) for 1 to 4 min. Platelet deposition was quantified by measuring the P-selectin (PS) and beta-thromboglobulin (betaTG) content of dissolved plasmin-digested thrombi using immunoenzymoassays; fibrin deposition was determined by measuring the D-dimer levels. These results were compared to those established by morphometrical analysis. Morphometric evaluation showed that fibrin deposition was maximum on TF by 1 min perfusion time. Platelets deposited subsequently and reached a maximum at 3 min. On collagen, platelets deposited directly on the collagen fibrils without detectable fibrin deposit. Platelet deposition increased from 1 to 4 min. Platelet deposition quantified by PS was correlated to the values obtained by morphometry (r = 0.72, r = 0.67, p <0.001, on TF and collagen, respectively). As compared to PS, betaTG measurements gave an underestimation of the size of the thrombus platelet number. Unfractionated heparin infused through a mixing device proximal to the perfusion chamber to obtain plasma concentrations of 0.5, 1 and 3 IU/ml, reduced fibrin deposition on TF-coated coverslips in a dose-dependent manner (77% reduction at 3 IU/ml, p <0.01), but had no significant effect on platelet deposition (33% at 3 IU/ml, p >0.05). In contrast, heparin had no effect on fibrin or platelet deposition on collagen-coated coverslips. Thus, a new quantitative and simple method for measuring platelet deposition in flowing blood has been developed and characterized. Utilizing this system, we have demonstrated that unfractionated heparin did not inhibit arterial thrombus formation either on procoagulant or on proaggregant surface.

Using a Vortex To Disaggregate Platelet Clumps

Clumping of platelets in blood anticoagulated with EDTA is a main cause of reports of falsely low platelet counts by automated analyzers. To obtain reliable platelet counts in such cases, laboratorians often collect and analyze a citrated or nonanticoagulated blood specimen. The patient thus is subjected to a second venous or capillary puncture, and the availability of results is delayed. Our data on an alternative approach suggest that reliable automated platelet counts can be obtained from many blood specimens that reveal microscopic platelet clumps after mixing them by vortex and rerunning the samples through the analyzer. Mixing the blood by vortex at the maximum possible speed for 1 to 2 minutes disaggregated platelet clumps completely in 44% of specimens and partially in another 49% of specimens.

Xylum CSA®: Automated System for Assessing Hemostasis in Simulated Vascular Flow

Nearly 20 years ago it became clear that analysis of hemostasis function in blood was affected by sample shear rate and anticoagulation (1). Yet, present technologies still perform hemostasis analysis on anticoagulated samples under static conditions. In 1984, Gorog and Ahmed developed a test system that analyzed hemostasis function of untreated whole blood under physiological flow (2), a technology potentially useful for assessing patients with bleeding disorders (2)(3). This approach has been automated in a user-friendly benchtop instrument, the Clot Signature Analyzer (CSA®; Xylum Corp.). From a single venipuncture, the CSA provides information on platelet adhesion induced by high shear, platelet aggregation, and coagulation attributable to humoral factors (4). Measurements are performed on untreated nonanticoagulated whole blood under the physiological conditions of nonrecirculating flow at 37 °C. The single-use, disposable CSA cassette consists of two perfusion channels, the “punch” and “collagen” channels. In each channel, blood is perfused in tubing under conditions simulating vascular flow. Blood is drawn into two 3-mL syringes, which are attached to luer fittings on the cassette. When the cassette is loaded onto the instrument, low-density oil is delivered into the syringes from a reservoir on the instrument. The imiscible oil rises in the syringes and displaces the blood, which then flows into perfusion lines maintained at 37 °C. The luminal pressure exerted by the flowing blood is measured continuously in both channels by sensors on the instrument. In the punch channel, vascular injury is simulated by piercing the …

Platelet activation in flowing blood passing growing arterial thrombi.

We investigated the combined effect of wall shear rate and immobilized collagen on platelet activation in flowing nonanticoagulated human blood. By combining an ex vivo model of thrombogenesis with flow cytometry, we showed that activated platelets can be detected in the bloodstream passing growing thrombi at a wall shear rate characteristic of moderately stenosed arteries (2600 s-1). The activation of the circulating platelets was clearly correlated with thrombus growth. Different antibodies against platelet activation-dependent surface markers had distinct sensitivity to the thrombotic process. alpha-Granule release detected by surface expression of CD62P seemed to be the most sensitive marker, as judged by both mean fluorescence intensity and fraction of platelets activated. The conformational change in glycoprotein IIb-IIIa, as detected by PAC-1, also seemed to be a sensitive marker and preceded binding of fibrinogen to activated glycoprotein IIb-IIIa, as detected by anti-fibrinogen. Large thrombi also elicited lysosome exocytosis, detected by surface expression of CD63. Finally, we observed a small decrease of glycoprotein Ib-IX expression, as detected by anti-CD42a. Thus, our study provides further information on the dynamics of platelet activation in relation to thrombus growth at arterial shear conditions in flowing nonanticoagulated human blood.

Relative involvement of GPIb/IX-vWF axis and GPIIb/IIIa in thrombus growth at high shear rates in the guinea pig.

The relative involvement of the glycoprotein (GP) Ib/IX-von Willebrand factor (vWF) axis and GPIIb/IIIa in thrombus growth at high shear rates was assessed and compared by testing the pharmacological effects of VCL, a recombinant GPIb-binding fragment of vWF (residues 504-728), aurintricarboxylic acid (ATA), which binds to the 509-695 disulfide loop of vWF, and lamifiban, a specific synthetic GPIIb/IIIa antagonist. In vivo, their effects were evaluated in guinea pig mesenteric arteries, in a model of a laser-induced cyclic thrombotic process, and ex vivo, at a shear rate of 1800 s(-1), in a capillary perfusion chamber model, in which collagen-adherent platelets are exposed to nonanticoagulated guinea pig blood. In vivo, VCL, ATA, and lamifiban administered 2 minutes after intimal injuries stopped thrombus growth, prevented the cyclic thrombotic process, and induced gradual thrombus dissolution. Ex vivo, at 1800 s(-1), collagen exposure to untreated blood for 2 minutes, 4 minutes, or two consecutive periods of 2 minutes each resulted in similar platelet adhesion, 56%, 59%, and 61%, respectively, with an average thrombus volume of 6, 19, and 17.5 microm3/microm2, respectively, without any fibrin formation. This indicated that the two consecutive perfusions did not affect the dynamic process of thrombus growth. When collagen-adherent platelets deposited after the first 2-minute perfusion were perfused for 2 minutes with VCL-, ATA-, or lamifiban-treated blood, thrombus growth was prevented and platelet adhesion remained unchanged, but fibrin formation increased on and around the predeposited platelets. These results suggest that both the GPIb/IX-vWF axis and GPIIb/IIIa are involved in in vivo platelet-to-platelet interactions at high shear rates in the guinea pig.

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.

Acute thrombogenicity of intact and injured natural blood conduits versus synthetic conduits: Neutrophil, platelet, and fibrin(ogen) adsorption under various shear-rate conditions

We investigated the acute thrombogenicity of synthetic arterial prostheses compared to biological arterial surfaces in contact with flowing nonanticoagulated blood. The acute events following blood/surface interactions were quantified using 51Cr-platelet deposition, 111In-neutrophil adhesion, and 125I-fibrin(ogen) adsorption on expanded polytetrafluoroethylene (ePTFE) synthetic arterial surfaces (Goretex and Impra) and on intact and injured biological arterial surfaces in ex vivo superfusion flow chambers at low (424/sec) and high (3397/sec) shear rates for 5 min at 37 degrees C. The hematological parameters were determined, and surface analysis was assessed by scanning electron microscopy. At low shear rate, the retention on intact arterial surfaces averaged 3.7 +/- 0.7 x 10(6) platelets/cm2, 26.5 +/- 4.2 x 10(3) neutrophils/cm2, and 10.7 +/- 2.2 cpm of fibrin(ogen)/cm2; retention remained statistically similar at the high shear rate on both Goretex and Impra ePTFE surfaces. In contrast, the deposition of platelets and neutrophils on injured arterial surfaces was significantly higher and increased with shear rate, although the significant increase in fibrin(ogen) adsorption was not influenced by the shear rate. At shear rates characterized by patent and stenosed arteries, ePTFE arterial prostheses demonstrated a low level of thrombogenicity compared to injured arteries. This favorable comparison can be considered as the first requirement for their successful use in arterial substitution.

The thrombomodulin/protein C/protein S anticoagulant pathway modulates the thrombogenic properties of the normal resting and stimulated endothelium.

We investigated the role of the thrombomodulin (TM/protein C/protein S anticoagulant pathway in modulating the thrombogenic properties of the endothelium. Endothelial cells (ECs) were placed in parallel-plate flow chambers and exposed to nonanticoagulated human blood at a venous wall shear rate (50 s-1). Fibrin deposition on resting ECs treated with a control IgG1 was negligible. In contrast, a significant amount of fibrin deposited when TM expression was specifically suppressed by > 95% by preincubating ECs with an anti-TM IgG1. Similarly, fibrin deposited on interleukin 1-stimulated ECs, but the fibrin deposition was further increased threefold with anti-TM IgG1. Comparable results were found when ECs were perfused at 650 s-1. When TM surface activity was enhanced by 150% by treating ECs with active phorbol ester (4-phorbol 12-myristate 13-acetate; PMA), the deposition of fibrin was 30% lower than on ECs not pretreated with PMA. Finally, fibrin deposition on stimulated ECs was significantly higher in 11 untreated patients with well-characterized deficiencies of protein C or S or heterozygous factor V Leiden mutation than in 11 healthy individuals, and it was significantly correlated to basal plasma levels of thrombin-antithrombin complexes. Thus, this study underlines the central role of the TM/protein C/protein S pathway in modulating the thrombogenic status of resting and stimulated ECs and indicates that basal coagulation system markers may be helpful in monitoring patients presenting a disorder of this anticoagulant pathway.

Endothelium-derived Nitric Oxide Decreases Polymorphonuclear Leukocyte Interaction with the Deeply Injured Arterial Wall under Intermediate and High Shear Conditions

Previous studies have shown that nitric oxide (NO) inhibits specific agonist-induced polymorphonuclear leukocyte (neutrophil) and platelet aggregation in vitro. However, the inhibitory effects of NO on neutrophil interaction with the deeply injured arterial wall under conditions of flow is unknown. Therefore, we investigated the influence of NO derived from the endothelium on neutrophil and platelet interactions with the downstream arterial media under controlled flow conditions. Porcine aortic media, simulating deep arterial wall injury, was exposed to flowing porcine non-anticoagulated arterial blood for 5 min at intermediate (1006 s(-1)) and high (3397 s(-1)) shear conditions, and deposition of radiolabeled neutrophils and platelets was quantified. Neutrophil deposition on the exposed arterial media was reduced, by more than 30%, by pretreatment of the endothelium with the physiological precursor of NO, L-arginine, from 84.1 +/- 13.7 to 57.4 +/- 7.2 x 10(3)/cm2 (p < 0.05) at 1006 s(-1), and from 99.3 +/- 9.8 to 65.5 +/- 8.7 x 10(3)/cm2 (p < 0.05) at 3397 s(-1) of shear rate, relative to control. Pretreatment of the endothelium with the inactive stereoisomer D-arginine had no effect on neutrophil deposition. These specific inhibitory effects of L-arginine were completely abolished by the inhibitor of NO synthesis, N(omega)-nitro-L-arginine methyl ester (L-NAME) at both shear rates. Endothelial pretreatment with D-arginine, or with L-arginine, in the absence or presence of L-NAME, did not significantly influence platelet interaction with the thrombogenic arterial media at intermediate and high shear rates. These results indicate that NO derived from the endothelium can modulate and has a greater influence on neutrophil, than on platelet, interaction with the injured arterial wall exposing the media under conditions of flow typical to moderately and severely stenosed arteries.

Comparativein VitroAnalysis of Topical Hemostatic Agents

Hemostatic agents are broadly utilized across the surgical specialties. While specific characteristics desirable for individual applications may vary, these agents generally are expected to aid the patient's coagulation system in the rapid development of an occlusive clot. Six commonly utilized hemostatic agents were quantitatively compared in terms of their ability to mediate platelet aggregation, deposition and activation, and initiate gross clot formation in a series ofin vitrotests. Three types of collagen sponges (Actifoam, Helistat, Instat), microfibrillar collagen (Avitene), a gelatin sponge (Gelfoam), and oxidized regenerated cellulose (Surgicel) were studied. Platelet aggregation: Citrated platelet rich plasma (PRP) was contacted with hemostatic agents both with and without thrombin in a stirred cuvette and the platelet count was measured over 5 min. Platelet deposition: To approximate arterial wounds, PRP was perfused in a controlled manner through hemostatic agents, and the effluent platelet count was measured over time. Platelet activation: ATP secretion from PRP contacted with hemostatic agents in a stirred cuvette was measured at 1 and 5 min. Clot formation: The clotting time of nonanticoagulated whole blood contacted with the hemostatic agents was measured. Materials which depleted platelets most rapidly and effectively in the perfusion system (Avitene, Helistat, and Actifoam) were also the most effective inducers of platelet aggregation and secretion. Clot formation (likely to be platelet dependent) was also more rapid in this group. An overall activity ranking combined the relative performance of each hemostatic agent on the various tests: Actifoam ∼ Avitene > Helistat >> Gelfoam > Instat > Surgicel. The activity ranking generally reflects the materials used in these agents (collagen > gelatin > oxidized regenerated cellulose), as well as their processing (chemical crosslinking in collagen sponges may lower activity). Suchin vitroassessment of the relative platelet and coagulation activities of hemostatic agents might serve as a useful screening tool before investigating properties which require more expensive animal or clinical testing.

Sustained inhibition of whole-blood clot procoagulant activity by inhibition of thrombus-associated factor Xa.

Progression of arterial thrombosis partly depends on thrombus-associated thrombin and activated factor X (Xa) activity. However, whether Xa or thrombin is the most appropriate target for inhibition of recurrent thrombosis is unknown. This study was designed to determine whether inhibition of Xa results in more sustained attenuation of thrombus-associated precoagulant activity than does inhibition of thrombin. Clots prepared ex vivo from human whole blood and pathological arterial thrombi from patients were preincubated in citrated plasma containing no inhibitor, 0.5 to 1 U/mL heparin, 0.5 to 1 mumol/L hirudin, 5 to 10 mumol/L tick anticoagulant peptide (TAP), 0.15 to 3 mumol/L tissue factor pathway inhibitor (TFPI), or a combination of 1 mumol/L hirudin and 10 mumol/L TAP for 2 hours. After preincubation the clots were removed from first-stage plasma, extensively washed in phosphate-buffered saline, and added to nonanticoagulated whole blood. Clots preincubated in plasma without inhibitors induced marked activation of the coagulation system in whole blood, as characterized by greater increases in the concentration of fibrinopeptide A (FPA) over 7 minutes than in blood without added clots (1522 +/- 568 compared with 117 +/- 170 ng/mL, P < .01). Preincubation of clots with heparin or hirudin did not attenuate the increases in FPA in wholeblood. In contrast, compared with incubation without an inhibitor, preincubation of clots with TAP or TFPI markedly attenuated the increases in FPA when clots were added to whole blood (551 +/- 316 and 508 +/- 208 ng/mL, respectively, P < .01). Similar results were obtained with arterial thrombi from patients. Inhibition of Xa but not of thrombin results in sustained attenuation of thrombus-associated procoagulant activity. Uninhibited thrombus-associated Xa activity may account for the increases in thrombin activity that are commonly observed in clinical trials after discontinuation of thrombin inhibitors.

Biocompatibility of sulphonated polyurethane surfaces

Surfaces of medical devices made of polymeric materials may promote thrombosis and inflammation. Therefore, in an attempt to produce surfaces which might diminish biomaterial-mediated thrombosis and inflammation, surface derivatization with 2-acrylamido-2-methylpropanesulphonic acid (AMPS®) was carried out. The derivatization procedure generates free radicals which initiate the copolymerization of AMPS monomers directly to a polyurethane surface. In an in vitro blood loop study using non-anticoagulated human blood, the resulting AMPS-derivatized material completely abrogates the generation of fibrinopeptide A, decreases the production of β-thromboglobulin and C3a, and decreases the adherence of platelets. The derivatized material also attracts fewer adherent neutrophils when implanted in mice. However, AMPS derivatization unexpectedly increases the recruitment of macrophages to implanted material and promotes the formation of adherent sleeve thrombi on central venous catheters indwelling in non-anticoagulated canine femoral veins. Thus, AMPS derivatization has highly variable effects on inflammatory and thrombotic systems. Further investigation is clearly required to determine the mechanisms underlying both desired and adverse effects.

In vitro Blood Compatibility Evaluation of Hollow Fibre Membrane Using a Controlled Flow System: A Comparative Study

A procedure has been established for the in vitro assessment of hollow fibre haemodialysis membranes. A 30 ml syringe containing 20 ml of fresh non-anticoagulated blood was mounted onto a non-pulsatile syringe pump and blood was perfused through minimodules constructed from 80 fibres retrieved from Cuprophan (Baxter ST15), cellulose acetate (M57-12, JMS Co Ltd, Hiroshima, Japan), and AN69HF (Filtral 20, Hospal, France) dialysers. Samples were collected before perfusion, 3, 6, 9 and 12 minutes. The modules were clamped vertically to minimise the effect of red cell pooling and the dialysate compartment was filled with 0.9% saline to minimise ultrafiltration. After sample processing, complement C3a, thrombin-antithrombin III complexes, prothrombin F1 + 2, and factor XII-like activity were evaluated. The results indicated that the system could discriminate between the membranes evaluated and therefore was a relevant procedure for the assessment of hollow fibre haemodialysis membranes.

VARIABLE INFLUENCE OF HEPARIN AND CONTRAST AGENTS ON PLATELET FUNCTION AS ASSESSED BY THE IN VITRO BLEEDING TIME

Both heparin and contrast agents have anticoagulant effects which are well-documented but their effects on platelets are not well-characterized. The purpose of the present study was to evaluate the sequential effects of heparin and then a contrast agent on platelet function during an angiographic procedure. Blood samples from 54 patients were obtained at baseline, after a 5000 unit bolus of heparin and after administration of a contrast agent (iohexol, n=30: diatrizoate, n=24) during angiography. The in vitro bleeding time (IVBT) was determined on nonanticoagulated whole blood using a hollow fiber device under physiological flow conditions. Mean IVBT at baseline was 3.6 ± 2.7 minutes and increased to 17.0 ± 12.3 minutes after heparin (p < 0.01). After heparin, 44.5% of the patients still had a normal IVBT (< 9.0 minutes), 11% of the patients had a moderately increased IVBT and the remaining patients had a large increase in their IVBT. When contrast was given (167 ± 52 mls) following heparin, mean IVBT was higher in those who received diatrizoate (23.3 ± 9.4 minutes) compared with iohexol (15.0 ± 10.9 minutes, p < 0.05). However, 15 patients (28%) continued to have a normal IVBT after contrast and of these 80% had received iohexol.

Procoagulant effect of incompatible platelet transfusions in alloimmunized refractory patients.

The clinical effectiveness of platelet transfusion in refractory patients is still a subject of debate. We have evaluated the possible hemostatic effect of platelet transfusion in 16 alloimmunized thrombocytopenic patients whose platelet counts were less than 20,000/microliters. Platelet concentrates were always obtained by apheresis procedures from incompatible donors. The posttransfusion platelet recovery was greater than 15% only in 3 cases. In the first 6 patients, measurements of bleeding time performed immediately before transfusion were in all cases longer than 30 min and did not change significantly 10 and 60 min after platelet transfusions. In all patients, ex vivo perfusion experiments with Baumgartner's platelet adhesion model, using native nonanticoagulated blood, were performed immediately before and 10 and 60 min after transfusion. No difference in platelet deposition onto the subendothelial surface was observed after platelet transfusion. Unexpectedly, the deposition of fibrin on the subendothelial surface was statistically augmented in the posttransfusion studies. Quantification of thrombin-antithrombin complexes (TAT) in plasma showed statistically significant elevations (p < 0.01) in the posttransfusion samples (31.9 +/- 12.6 vs. baseline 5.8 +/- 1.7 ng/ml), not justified by TAT levels in the transfused material (2.3 +/- 0.17 ng/ml). Transfusion of incompatible platelets to refractory patients may activate coagulation mechanisms in the absence of an increase in peripheral platelet count.

GUINEA PIG BLOOD: A MODEL FOR THE PHARMACOLOGIC MODULATION OF THE GPIb/IX-VWF AXIS

Antithrombotic activity of two recombinant GPIb-binding fragments of vWF, RG12986 (residues 445-733), and VCL (residues 504-728), were assessed in an ex vivo capillary perfusion chamber exposing human type III collagen to native nonanticoagulated guinea pig blood. Platelet adhesion and thrombus formation were evaluated by computer assisted morphometry for two shear rates (650 and 1800 s −1) and for two perfusion times (1.5 and 4 min). At 1800 s −1 and 4 min of perfusion, platelet adhesion decreased from 63 ± 7% for control, to 46 ± 4% for 20 mg/kg RG12986, and to 29 ± 5% for 4 mg/kg VCL, and the mean thrombus height dropped from 40 ± 8 μm to 24 ± 3 μm and 7.5 ± 1 μm, respectively. The two doses did not change bleeding time values. Our results suggest that guinea pig blood and the circular perfusion chamber represent a good model for the evaluation of limited amount of GPIb/IX-vWF axis inhibitors.

Cyclosporine-induced detachment of vascular endothelial cells initiates the intrinsic coagulation system in plasma and whole blood

Cyclosporine A (CsA) is supposed to alter the metabolism of vascular endothelial cells, leading to a prothrombotic state. We examined by which mechanism human umbilical vein endothelial cells (HUVECs) treated with CsA would promote coagulation in human plasma and in whole blood. Treatment of HUVECs with CsA at concentrations clinically used led to dose-dependent cell detachment, with the subsequent exposure of the highly procoagulant connective tissue. As determined by scanning electron microscopy, cell counting of detached and adherent cells, and antigenic measurement of collagen exposure, HUVECs treated with 0.4 μg/ml CsA (or more) for 4 days exhibited significant amounts of subendothelial areas. On CsA-treated HUVEC monolayers, the clotting time of recalcified citrated plateletrich plasma (PRP), but not platelet-poor plasma (PPP), was dose-dependently shortened. Likewise, the onset of thrombin generation was significantly earlier. Except at a high concentration of 8.0 μg/ml CsA, there was no procoagulant effect when PPP was used. To investigate CsA-treated HUVECs in whole blood, cells were cultivated on globular microcarriers and were incubated with nonanticoagulated whole blood. When untreated cells were used, generation of factor Xa, thrombin, and kallikrein was completely suppressed for 30 minutes. HUVEC beads treated with 0.4 and 0.8 μg/ml CsA, however, led to a dose-dependent generation of all three coagulation factors, with peak values at 2.5 to 5 minutes. Extrinsic activation was excluded, since CsA treatment did not induce tissue factor activity in HUVECs. Furthermore, the thrombomodulin activity of HUVECs was not altered by CsA. In conclusion, treatment of HUVECs with CsA for 4 days at concentrations clinically used leads to the exposure of subendothelial areas that induce activation of the intrinsic coagulation in recalcified PRP and nonanticoagulated whole blood.

Effect of iodixanol on in vitro bleeding time

Both monomeric and dimeric ionic radio contrast media (CM) have been shown to be more anticoagulant than nonionic monomeric CM. Iodixanol is a relatively new nonionic dimeric contrast medium. We investigated whether the dimeric structure of iodixanol would produce the same level of anticoagulation that has been observed using nonionic monomeric CM. We used a global screening device that operates under physiologic flow conditions to monitor the effects of iodixanol on in vitro bleeding time (IVBT). This flow dynamic technique perfuses nonanticoagulated whole blood through a hollow fiber device. A leak in the fiber is created by a precision needle, and the resulting pressure fluctuations within the fiber are monitored to examine the ability of the hemostatic system to close the leak by forming a stable platelet plug. In 20 donors, iodixanol (25% CM/blood, v/v) was shown to lengthen the mean IVBT (18.74 min) compared with the normal blood mean IVBT (4.24 min). The addition of dimeric iodixanol to normal blood affects the IVBT in the same manner as nonionic monomeric CM (ioversol, iopamidol, and iohexol).