Single ascending oral dose pharmacokinetics and pharmacodynamics study of EV-077: the specific inhibitor of prostanoid- and isoprostane-induced cellular activation.

PURPOSE: This study was performed to determine the oral pharmacokinetics (PK) of EV-077 and its effects on pharmacodynamic (PD) markers. EV-077 blocks prostanoid-induced and isoprostane-induced cellular activation, and is in development for the treatment of vascular inflammation and associated complications of type-2 diabetes.. METHODS: This single-ascending-dose mono-centre study was randomised, placebo-controlled, and double-blinded within each dose group. Seven EV-077 doses were administered sequentially as an oral solution: 0.0125, 0.125, 0.375, 0.75, 1.25, 1.875 and 2.5 mg/kg body weight. PK, platelet aggregation, bleeding time and safety parameters were measured. Seven to eight healthy male subjects were dosed per group: five to six subjects received EV-077 and two subjects received placebo. RESULTS: Tmax was reached rapidly between 0.5 h and 1.0 h. Both Cmax and AUC increased linearly with the dose. The apparent terminal half-life (t½z) increased with the dose, most likely reflecting the increasing last quantifiable concentration with increasing dose; at 2.5 mg/kg, it was 2.7-6.9 h. Measurement of platelet aggregation showed no effect at 0.0125 mg/kg, and a full and reversible inhibition at doses of 0.125-2.5 mg/kg. The average bleeding time was dose-dependently prolonged, but was always below 9 min. The PK/PD profile showed that at plasma concentrations above 20 ng/ml, EV-077 platelet aggregation was completely inhibited (>90 %). All tested doses were well tolerated. CONCLUSIONS: Orally administered EV-077 was well tolerated, readily absorbed, reached Cmax within 1 h, with a linear PK based on Cmax and AUC. The inhibition of platelet aggregation was complete and reversible at doses of 0.125 mg/kg and higher, and average bleeding time was below 9 min.

Role of factor VIII-von Willebrand factor and fibronectin in the interaction of platelets in flowing blood with monomeric and fibrillar human collagen types I and III.

Platelet adhesion to monomeric collagen types I and III, which were purified from human umbilical arteries, was studied in a perfusion chamber under well defined flow conditions. For this purpose, glass coverslips were coated with 20-30 micrograms/cm2 of collagen types I and III by spraying a solution of these collagens with a retouching air brush. Platelet deposition increased with the time of perfusion. Adhesion to both collagen types was similar in the first 3 min, but increased platelet deposition occurred on collagen type III after 3 min due to thrombus formation. Adhesion at a shear rate of 800 s-1 was strongly impaired with plasma of a patient with von Willebrand's disease or with fibronectin-free plasma. Addition of purified fibronectin to fibronectin-free plasma restored adhesion to the level obtained with normal plasma. Platelet deposition in normal plasma increased with increasing shear rates. Platelet deposition in VWD-plasma was normal at 490 s-1, but there was no increase at higher shear rates. Platelet deposition in fibronectin-free plasma was diminished at all shear rates studied from 490 to 1,300 s-1. Perfusion with a human albumin solution (HAS) to which purified Factor VIII-von Willebrand factor complex (FVIII-VWF) and fibronectin had been added gave similar platelet deposition as with normal plasma. Preincubation of collagen with FVIII-VWF and perfusion with HAS containing fibronectin, or, conversely, preincubation with fibronectin and perfusion with HAS containing FVIII-VWF, also resulted in adhesion similar to that observed in normal plasma. Similar adhesion was also observed after preincubation with both FVIII-VWF and fibronectin and subsequent perfusion with HAS alone. Sequential preincubations with first FVIII-VWF and then fibronectin, or with first fibronectin and then FVIII-VWF followed by perfusion with HAS, also gave a similar adhesion as observed with normal plasma. These data indicate that platelet adhesion to monomeric collagen types I and III is dependent on both FVIII-VWF and fibronectin. FVIII-VWF is only required at relatively high shear rates; fibronectin also at relatively low shear rates. Their complementary role in platelet adhesion suggests separate binding sites for FVIII-VWF and fibronectin on collagen. Platelet deposition on performed fibrils of collagen types I and III was also studied. Initial adhesion expressed as percentage surface coverage was similar to that found with monomeric collagen, but thrombus formation was much enhanced. Adhesion on fibrillar collagen at 800 s(-1) was impaired in VWD-plasma and fibronectin-free plasma, and was restored by addition of purified fibronectin to fibronectin-free plasma. When perfusions were performed with HAS, only addition of FVIII-VWF was required for optimal adhesion to fibrillar collagen; addition of fibronectin had no effect. These data are in contrast to the studies with monomeric collagens described above, in which the addition of both FVIII-VWF and fibronectin was required. These data are also in contrast to the observation that in plasma both FVIII-VWF and fibronectin are required for optimal adhesion to fibrillar collagen.

Functional domains on von Willebrand factor. Recognition of discrete tryptic fragments by monoclonal antibodies that inhibit interaction of von Willebrand factor with platelets and with collagen.

We have identified two functional domains on the von Willebrand factor (VWF) moiety of the Factor VIII-von Willebrand factor complex (FVIII-VWF), one interacting with blood platelets, and one interacting with vessel wall collagens, by means of two monoclonal antibodies directed against the VWF molecule, CLB-RAg 35 and CLB-RAg 201. The monoclonal antibody CLB-RAg 35 inhibited virtually all platelet adherence to artery subendothelium and to purified vessel wall collagen type III, at relatively high wall shear rates. CLB-RAg 35 also inhibited the ristocetin-induced platelet aggregation and the binding of FVIII-VWF to the platelet in the presence of ristocetin but did not affect the binding of FVIII-VWF to collagen. The monoclonal antibody CLB-RAg 201 inhibited the binding of FVIII-VWF to purified vessel wall collagen type I and III and all platelet adherence to collagen type III and the platelet adherence to subendothelium that was mediated by FVIII-VWF in plasma. The two functional domains on FVIII-VWF that were recognized by CLB-RAg 35 and CLB-RAg 201 were identified by means of immunoprecipitation studies of trypsin-digested FVIII-VWF. The domains resided on different polypeptide fragments, with a Mr of 48,000 for the collagen binding domain and a Mr of 116,000 for the platelet binding domain. The 116,000-mol wt fragment consisted of subunits of 52,000/56,000 mol wt and 14,000 mol wt after reduction. The 52,000/56,000-mol wt subunits possessed the epitope for CLB-RAg 35.

Axial dependence of collagen-induced thrombus formation in flowing non-anticoagulated human blood. Anti-platelet drugs impair thrombus growth and increase platelet-collagen adhesion.

Platelet thrombus formation on collagen fibrils is most pronounced at the upstream end of the surface, and it gradually decreases along the axis in parallel with the direction of the blood flow. This phenomenon, known as axial dependent platelet thrombus formation, is explained by the balance of the platelet supply to the surface and the consumption of platelets by growing thrombi. In the present study we have affected this balance by (A) inhibiting the growth of platelet thrombi by aspirin (ASA) or clopidogrel, and thus increasing the platelet concentration at the surface, and by (B) utilising blood from cigarette smokers, who have enhanced thrombus formation immediately after smoking, and thus decreasing the platelet concentration at the surface. Thrombus formation in non-anticoagulated blood was triggered by collagen fibrils positioned in a parallel-plate perfusion chamber at a wall shear rate of 2600 s-1 which is characteristic for moderately stenosed arteries. Morphometrical assessment of thrombus formation was performed at axial positions of 1 and 13 mm downstream to the blood flow inlet at the collagen surface. Platelet-collagen adhesion and thrombus volume in blood from nonsmokers were decreased at the downstream location by 39% (p < or = 0.0001) and by 60% (p < or = 0.0001), respectively. However, increasing the platelet concentration at the surface by partially inhibiting the thrombus growth by ASA or clopidogrel, reduced substantially the axial decrease in platelet adhesion and thrombus volume.(ABSTRACT TRUNCATED AT 250 WORDS)

Collagen induced thrombus formation at the apex of eccentric stenoses--a time course study with non-anticoagulated human blood.

Atherosclerotic plaque rupture may trigger the formation of mural thrombus. This thrombus formation is apparently affected by very high and complex shear conditions introduced by the luminal narrowing (stenosis) of the atheroma. To study the impact of such blood flow behaviour on thrombus formation we employed a model system where collagen-induced thrombogenesis is studied at the apex of well-defined eccentric stenoses. Thrombus formation in non-anticoagulated human blood drawn directly from an antecubital vein over the collagen coated stenosis apex for periods of 0.5, 1, 3 or 5 min was quantified by morphometry. The stenoses reduced the cross-sectional area of the blood flow channel by 60, 80 and 89%, which corresponded to apex wall shear rates of 2600, 10,500 and 32,000 s-1, respectively. Platelet-collagen adhesion decreased by increasing shear at the stenosis apex. The corresponding adhesion rates were highest at 1 min, then they gradually decreased upon prolongation of the perfusion time. The platelet thrombus volume increased in concert with increasing shear rate up to 10,500 s-1, whereas, at 32,000 s-1, the volume wa decreased. The corresponding growth rates and rates of thrombus occlusion at the apex levelled off at 3 min. Significant fibrin deposition was not observed before 3 min, and was most pronounced at 10,500 and 32,000 s-1. The plasma levels of fibrinopeptide A and beta-thromboglobulin increased in concert with increasing shear and perfusion time, particularly at the two highest shear conditions. Thus, hallmarks of thrombus formation at these stenoses with increasing shear are decreased platelet-collagen adhesion, and increased platelet-platelet interaction and fibrin deposition. A fibrin tail downstream to the collagen-attached platelet thrombus is regularly observed when thrombus occlusion exceeds 40%. However, the reduced thrombus growth at the most occlusive stenosis (89%) is presumably due to the high shear stresses which may reduce the rate of platelet incorporation into the thrombus and/or tear off thrombus fragments.

Upstream thrombus growth impairs downstream thrombogenesis in non-anticoagulated blood: effect of procoagulant artery subendothelium and non-procoagulant collagen.

In the present experiments we have investigated the influence of wall shear rate and axial position on platelet and fibrin deposition which results when flowing human non-anticoagulated blood is exposed to either non-procoagulant fibrillar collagen (human type III) or procoagulant subendothelium (rabbit aorta). Platelet adhesion, thrombus volume and fibrin deposition were morphometrically evaluated at axial positions of 1 and 13 mm following perfusions for 5 min at shear rates of 100, 650 and 2,600 s-1. An axially-dependent decrease of platelet adhesion (34-57%, p less than 0.01-0.05) and thrombus volume (57-80%, p less than 0.05) was observed on collagen at all shear rates. On subendothelium, an axially-dependent decrease was observed for platelet adhesion only at 100 s-1 (29%; p less than 0.01) and for thrombus volume at shear rates of 650 s-1 and above (49-58%, p less than 0.01). Deposition of fibrin on subendothelium was axially decreased (16-42%, p less than 0.05) at all shear rates, while no significant axial differences were seen on collagen. However, substantially more fibrin was deposited on the subendothelium (p less than 0.05), and the upstream platelet adhesion and thrombus volume were lower than on collagen (p less than 0.05) at 100 s-1 and 650 s-1. The axially-dependent phenomena on the two surfaces are consistent with the concept of rapid-growing upstream thrombi which deplete the blood layer streaming adjacent ot the surface of platelets, leading to decreased platelet deposition further downstream.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth and stability of thrombi in flowing citrated blood: assessment of platelet-surface interactions with computer-assisted morphometry.

The differential quantitation of platelet deposition in perfusion studies is a major problem. We report on methods to prepare semithin sections of platelet deposits on collagen coated on glass and plastic cover slips, to study growth and stability of thrombi in three dimensions, and the development of a computer-assisted differential quantitation of platelet-collagen interactions. The interactions were quantified as percentage of the surface covered with platelets (platelet adhesion), thrombus height, thrombus density and thrombus area per unit sectional length, respectively. Cover slips coated with fibrillar equine collagen in parallel-plate perfusion chambers were exposed to flowing citrated blood at shear rates ranging from 200 to 2,600 s-1. Thrombi, partially enmeshed in the collagen meshwork, prevailed on the surface at all shear rates. Maximal platelet adhesion and thrombus density were seen at greater than 5 micrograms/cm2 collagen, while thrombus area and height were maximal at greater than 10 micrograms/cm2. The volume of the thrombi appeared correlated to the number of deposited platelets (r = 0.92). En face preparations showed deposits of platelet islands which grew in diameter with time, particularly in the direction of the blood flow, becoming progressively confluent. Sections cut parallel to the direction of the blood stream indicated that this growth pattern was at least partially caused by thrombi bent in the direction of the blood flow.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the haemostatic defect in a complicated syndrome. An inverse Scott syndrome platelet membrane abnormality?

The Stormorken syndrome is a multifacetted syndrome including a bleeding tendency. No deviations were found in the coagulation- or fibrinolytic systems. Platelet number was low normal, and size abnormal, whereas EM findings were unremarkable. Survival time was half normal. Clot retraction was initially rapid, but clearly decreased, whereas prothrombin consumption was also initially rapid, but complete. Membrane GP's were normal, so was AA metabolism, PI-cycle, granule storage and secretion, and c-AMP function, whereas 5-HT uptake and storage was decreased. Optical platelet aggregation was low normal with all physiological agonists. The only clearly abnormal finding was that coagulant activity was present on non stimulated platelets at the same level as kaolin-stimulated normal platelets. This indicated a platelet abnormality which should lead to a thrombogenic, not to a haemorrhagic trait. This paradox may have its origin in rheology, because when challenged with in vivo shear rates in an ex vivo perfusion chamber, platelet cohesion was abnormally low. Further studies to better delineate the membrane abnormality are underway.

A comparative study of the anticoagulant and anti-thrombotic effects of unfractionated heparin and a low molecular weight heparin (Fraxiparine) in an experimental model of human venous thrombosis.

We have compared the anticoagulant and the antithrombotic effects of unfractionated heparin (Calciparine) and low molecular weight heparin (Fraxiparine) in an experimental human venous thrombosis model. One single subcutaneous injection of Calciparine or Fraxiparine was administered to healthy male volunteers at one month interval in a randomised and cross-over design. Ten subjects received doses used in man for preventing venous thrombosis (5,000 IU and 3,075 IU, respectively), and seven other subjects received curative doses (12,500 IU and 6,150 IU, respectively). Thrombus formation was measured 3 h and 8 h after drug administration. Non-anticoagulated human blood was drawn for 5 min directly from an antecubital vein over confluent cultured endothelial cells positioned in a parallel-plate perfusion chamber. The cells were previously stimulated for 4 h with lipopolysaccharides (10 micrograms/ml) and interleukin 1 beta (50 U/ml), resulting in optimal expression of biological active tissue factor. The wall shear rate at the cell surface was 50 s-1 and mimicked venous blood flow conditions. Immunologically quantified fibrin deposition on the stimulated cells was reduced only by curative doses of Calciparine and Fraxiparine at 3 h (3.4 +/- 0.8 versus 1.0 +/- 0.2 micrograms/cm/ and 2.6 +/- 0.8 versus 1.0 +/- 0.1 micrograms/cm2, respectively, p < or = 0.05). The influence of Calciparine and Fraxiparine on the formation of thrombin and fibrin was determined by measuring the plasma levels of thrombin-antithrombin III complexes and fibrinopeptide A (FPA) in blood samples collected distally to the perfusion chamber. The generation of these markers was significantly inhibited (50-83%) by both prophylactic and curative doses of Calciparine and Fraxiparine (p < or = 0.05). However, Fraxiparine still significantly inhibited the thrombin and fibrin generation at 8 h (p < or = 0.05), whereas Calciparine did not. The antithrombotic effects of both heparins were correlated with their plasma activities as measured by the antifactor Xa or the antithrombin assays. Thus, it appears in this model that Calciparine and Fraxiparine produce comparable antithrombotic effects at clinically comparable doses. However Fraxiparine has a longer-lasting anticoagulant activity than Calciparine. These results are in good agreement with clinical observations in man, and thus in favour of our model of human venous thrombogenesis for further studies of antithrombotic molecules.

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

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

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

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

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.

Comparison of platelet interaction with subendothelium of human renal and umbilical arteries and the extracellular matrix produced by human venous endothelial cells.

Platelet interaction with subendothelium of human renal and umbilical arteries and with the extracellular matrix produced by cultured human venous endothelial cells was compared in flowing citrated blood by using an annular and a rectangular perfusion chamber. The renal arteries were post mortem specimens from adults showing, often pronounced, intimal fibrosis, whereas the umbilical arteries had well organized parallel arranged smooth muscle cells, without elastic membranes. The extracellular matrix obtained after removal of endothelial cells with Triton X-100 was homogenously attached to its substratum. Significantly more platelets adhered to the extracellular matrix than to the subendothelia. This discrepancy was most pronounced in reconstituted blood with plasma from a patient with homozygous severe von Willebrand's disease (subtype III). No differences in platelet adherence and platelet aggregate formation were noted between the subendothelia. Platelet aggregate formation was poor on all surfaces. These data indicate that the extracellular matrix produced by endothelial cells is at least as reactive for the interaction with platelets as subendothelium, probably partly synthesized by smooth muscle cells.

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.

Association between bleeding time and platelet adherence to artery subendothelium.

The efficacy of five different factor VIII-von Willebrand factor (FVIII-VWF) preparations in mediating adherence of blood platelets to damaged vessel walls was tested in an annular perfusion chamber utilizing human arteries and reconstituted blood. FVIII-VWF-purified by Sepharose CL-4B chromatography and von Willebrand factor prepared from this preparation by dissociation with 0.25 M CaCl2 followed by Sepharose CL-6B chromatography were equally effective in mediating platelet adherence as FVIII-VWF in cryoprecipitate and in plasma from normal subjects. A commercial concentrate of FVIII-VWF (Hemofil, Hyland) used for the treatment of haemophiliacs did not mediate platelet adherence at normal levels of FVIII-VWF related properties. A recently developed high-purity FVIII-VWF preparation (Concentrate II) containing multimers of high molecular weight normalized the platelet adherence. Platelet adherence in plasma obtained from two patients with von Willebrand's disease (VWD) was impaired, but plasma samples obtained following treatment with Concentrate II mediated normal platelet adherence. The normalization of platelet adherence paralleled the normalization of the bleeding time. This platelet adherence assay offers an inexpensive and efficient in vitro tool to test the efficacy of FVIII-VWF preparations designed for VWD patients. Preparations such as cryoprecipitate and Concentrate II mediated the platelet adherence and normalized the bleeding time. The commercial preparation did not mediate platelet adherence and had no effect on the bleeding time.

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)

Relationship between endothelial tissue factor and thrombogenesis under blood flow conditions.

We have evaluated the relationship between the level of tissue factor (TF) expression by stimulated endothelial cells and thrombus formation under blood flow conditions. Cultures of human umbilical venous endothelial cells (HUVECs) were treated in order to express different levels of TF activity. They were stimulated for 4 h with either I) lipopolysaccharides (LPS, 10 micrograms/ml), II) recombinant interleukin 1 beta (IL1 beta, 50 UI/ml) or III) simultaneously with LPS and IL1 beta (LPS+IL1 beta). TF activity was low on confluent HUVECs or on the corresponding extracellular-matrix (ECM prepared by exposure of HUVECs to 0.1 N NH4OH). In contrast, it was high when HUVECs were stimulated with LPS or IL1 beta, and significantly higher (p < 0.05) with LPS+IL1 beta. The TF activity associated with the stimulated ECM was 2-fold higher (p < 0.05) than that expressed on the luminal surface of the stimulated HUVECs, irrespective of the agonist or combination of agonists used. These surfaces were exposed to non-anticoagulated human blood at a venous (50 s-1) and an arterial (650 s-1) wall shear rate in parallel-plate perfusion chambers for 5 min. Thrombus formation was morphologically quantified by measuring the deposition of platelets and fibrin. Fibrin deposition was also immunologically quantified. Fibrin deposition was related to the level of TF expression. Non-stimulated HUVECs and corresponding ECMs were not thrombogenic. The luminal surface of HUVECs stimulated with LPS or IL1 beta alone expressed low levels of TF activity and was a poor inducer of platelet deposition and fibrin deposition (< 15%) at 50 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)

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

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

Role of platelet membrane glycoproteins and von Willebrand factor in adhesion of platelets to subendothelium and collagen.

Development of in vitro perfusion systems and quantitative methods to evaluate various aspects of platelet-surface interactions have helped to elucidate some complex interactions in platelet adhesion and platelet-platelet cohesion. The importance of platelet GPs in these interactions was soon evident, and rapid progress in biochemistry and immunology has made it possible to study the roles of these GPs in more detail. From our studies, which made use of platelets with GP deficiencies, MAbs, purified vWF, proteolytic fragments of vWF, a synthetic peptide, and in vitro perfusion systems, it appeared that GPIb and GPIIb/IIIa function as receptors for vWF, which binds platelets to connective tissue at sites of vascular lesions (FIGURE 8). More detailed information about these interactions is expected in the near future when bioengineered products are available for testing in flow models and in other experimental approaches.

Methods and models to evaluate shear-dependent and surface reactivity-dependent antithrombotic efficacy.

The purpose of the present communication is to evaluate the importance of blood flow and surface reactivity for measurement of antithrombotic drug activity or efficacy in selected model systems of thrombus formation. Such information is essential for proper evaluation of antithrombotic drug profiles. The continuous development of flow-dependent thrombosis models for in vitro (anticoagulated blood) and ex vivo (native blood) studies and their application in in vivo animal models from the early 1970s and onwards are briefly considered. Central to this process was the development of various types of perfusion chambers in which a thrombogenic surface is exposed to flowing blood. Such perfusion chambers have been inserted into arteriovenous (AV) shunts in baboon, pig, dog, and rabbit. These approaches have allowed reproducible testing of traditional and novel experimental antithrombotic drugs, and studies on novel drug strategies under well-defined shear conditions and surface reactivity. Shear-dependent antithrombotic efficacy in these models is observed with anticoagulants such as unfractionated heparin, low-molecular weight heparins, or selective inhibitors of thrombin, Factor Xa, or Factor VIIa. However, the degree of shear dependency depends on the nature of the thrombogenic surface, e.g., the inhibition is more pronounced on a tissue factor (TF)-rich surface than on a collagen-rich surface, particularly at venous or low arterial shear. Platelet antagonists such as the COX-1 inhibitor aspirin, inhibitors of thromboxane A2 (TxA2) synthetase, the TxA2 platelet receptor, and of von Willebrand factor (vWf) are shear dependent also, being more efficient at high arterial shear. In contrast, the platelet ADP antagonist clopidogrel, or antagonists to the active platelet membrane glycoprotein IIb-IIIa complex (GPIIb-IIIa) are shear independent. At extremely high arterial shear, which activates platelets and elicit aggregates of circulating platelets, aspirin looses its antithrombotic effect, whereas ADP and GPIIb-IIIa antagonists still interrupt thrombus formation. In general, results obtained with these models mimic and predict antithrombotic efficacy in man when comparison is possible. Information on antithrombotic efficacy in flow devices with various thrombogenic surfaces is now sufficiently available to suggest recommendations for experimental conditions, particularly with regard to blood flow and reactive surfaces.