Rabbit Aorta Subendothelium Research Articles

Effects of the selective inhibition of platelet thromboxane synthesis on the platelet-subendothelium interaction.

1. Drugs that inhibit TxA(2) synthesis are used to reduce platelet aggregation. The aim of this study was to compare the effects of a cyclo-oxygenase (COX) inhibitor (acetylsalicylic acid, ASA), a thromboxane synthetase (TxS) inhibitor (dazoxiben) and a dual TxS inhibitor and TxA(2) receptor blocker (DT-TX 30) on platelet aggregation and the platelet-subendothelium interaction in flow conditions. 2. The techniques used in this in vitro study were platelet aggregometry in whole blood, and measurement of platelet thromboxane B(2) and prostaglandin E(2) production and leucocyte production of 6-keto-PGF(1alpha). The platelet-subendothelium interaction was evaluated in rabbit aorta subendothelium preparations exposed to flowing blood at a shear stress of 800 s(-1). Morphometric methods were used to calculate the percentage of subendothelium occupied by platelets. 3. The 50% inhibitory concentration (IC(50)) of DT-TX 30 in whole blood was in the range of 10(-7) micro M (induced with collagen or arachidonic acid) to 10(-5) micro M (induced with thrombin) or 10(-4) (induced with ADP). IC(50) values under all experimental conditions were lower with DT-TX 30 than with ASA. For thromboxane B(2) the IC(50) were: ASA 0.84+/-0.05 micro M, dazoxiben 765+/-54 micro M, DT-TX 30 8.54+/-0.60 micro M. Prostaglandin E(2) was inhibited only by ASA (IC(50) 1.21+/-0.08 micro M). Leucocyte 6-keto-PGF(1alpha) was inhibited by ASA (IC(50) 6.58+/-0.76 micro M) and increased by dazoxiben and DT-TX 30. The greatest reduction in percentage subendothelial surface occupied by platelets after blood perfusion was seen after treatment with DT-TX 30 in the range of concentrations that inhibited collagen-induced platelet aggregation (control group: 31.20+/-3.8%, DT-TX 30 at 0.1 micro M: 10.71+/-0.55%, at 1.0 micro M: 6.53+/-0.44%, at 5.0 micro M; 1.48+/-0.07%). All three drugs reduced thrombus formation, although ASA (unlike dazoxiben or DT-TX 30) increased the percentage surface occupied by adhesions. 4. In conclusion, the effect of specific blockage of TxS together with blockage of membrane receptors for TxA(2) can surpass the effect of ASA in inhibiting the platelet-subendothelium interaction in flow conditions.

An intermediate-purity factor VIII concentrate supports platelet adhesion under flow conditions.

Von Willebrand factor (vWF) binds to platelets and mediates platelet adhesion to subendothelium to support haemostasis. Factor VIII concentrates containing vWF have been recommended for treatment of bleeding episodes in von Willebrand disease (vWD) types 2 and 3. Their clinical efficacy in normalizing FVIII coagulant levels, shortening the bleeding time, stopping or preventing clinical bleeding and safety have been tested. However, the basic mechanisms of their effects on haemostasis have not been fully characterized. We have analysed the ability of vWF present in an intermediate-purity factor VIII concentrate (Haemate-P, Centeon) to bind to platelets (ultrastructural studies) and to support platelet adhesion under flow conditions (perfusion studies). For this purpose, Haemate-P or cryoprecipitate was added to washed platelet suspensions, or to vWF-depleted reconstituted healthy anticoagulated blood. Immunoelectron microscopy (IEM) revealed vWF arrangements on platelet surfaces which have been exposed to mixtures of the vWF-rich concentrate plus ristocetin. vWF levels in perfusates were confirmed by determination of ristocetin co-factor and vWF-antigen. Baumgartner's perfusion method and computer-assisted morphometry were used to evaluate platelet adhesion of the perfusates onto everted rabbit aorta subendothelium under standardized conditions (shear rate, 800 s(-1) , 10 min, 37 °C). vWF-depleted perfusates showed 15.8% (SEM 1.7) total covered surface (CS) with platelets. An increase in CS resulted when 0.40 or 0.80 IU vWF mL(-1) from Haemate-P (30.1%, SEM 3.0, P<0.05; 39.4%, SEM 3.1, P<0.008, respectively) were added. A similar increase was observed when cryoprecipitate was added to perfusates (28.6%, SEM 2.4, P<0.05 for 0.40 IU vWF mL(-1) ; 27.2%, SEM 2.9, P<0.01 for 0.80 IU vWF mL(-1) ). IEM confirmed that vWF from concentrates binds to platelets. Furthermore, perfusion studies revealed that the fractionated vWF supports platelet adhesion, thus providing experimental evidence of the therapeutic benefits exerted by Haemate-P.

Thrombin plays a key role in late platelet thrombus growth and/or stability. Effect of a specific thrombin inhibitor on thrombogenesis induced by aortic subendothelium exposed to flowing rabbit blood.

Thrombin is involved in the pathogenesis of venous and arterial thrombosis. This study addressed the question of the relative importance of thrombin in the early and late phases of thrombogenesis. The effect of Ro 46-6240 (1.43 mg/kg bolus and 0.1 mg/kg per minute i.v.), a novel, selective thrombin inhibitor on thrombogenesis induced by rabbit aorta subendothelium, was measured ex vivo in a perfusion chamber model after a short (5-minute) and long (30-minute) exposure time to rabbit native blood. The role of the perfusion time was assessed at shear rates of 100/s, 650/s, and 2600/s. These shear rates mimic blood flow conditions found in veins, arteries, and small or stenosed arteries, respectively. Fibrin deposition and platelet thrombus formation on subendothelium were evaluated by microscopic morphometry. In the presence of Ro 46-6240, fibrin deposition was abolished at both perfusion times and at all shear rates. In the 5-minute experiments, thrombus height was reduced by Ro 46-6240 at shear rates of 100/s (85%) and 650/s (35%) but not at a shear rate of 2600/s, whereas thrombus area was not affected at any shear rate. In contrast, both thrombus height and thrombus area were reduced (60% to 90%) by Ro 46-6240 in the 30-minute perfusion groups at all wall shear rates. The antithrombotic effect of Ro 46-6240 after 30-minute perfusion was confirmed by the minimal increase in the pressure difference between the entrance and the exit of the perfusion chamber compared with the control groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for thrombin binding to dermatan sulphate sites in the rabbit aorta subendothelium in vitro

Evidence for thrombin binding to dermatan sulphate sites in the rabbit aorta subendothelium in vitro

Evidence for thrombin binding to dermatan sulphate sites in the rabbit aorta subendothelium in vitro

The proposal that thrombin binds to dermatan sulphate chains of extracellular proteoglycans has been examined directly using the subendothelium of the rabbit aorta. Freshly excised aortas were de-endothelialized by balloon catheter in vitro and then incubated with 125I-thrombin to allow adsorption of 20-30 fmol of thrombin/cm2. Pretreatment of the subendothelium with FPR-thrombin or chondroitinase ABC partially inhibited thrombin binding, each by approximately 40-45%. The addition of dermatan sulphate inhibited, competitively, up to 50% of thrombin from binding to the subendothelium whereas chondroitin-4 or -6 sulphates had little or no effect. By contrast, protamine inhibited 90% of FPR-thrombin binding. Of subendothelium-bound thrombin, chondroitinase ABC released only a small proportion (3-12%) of bound thrombin but up to 44% of bound FPR-thrombin. It is concluded that, when 125I-thrombin is bound in vitro at a concentration of < 30 fmol/cm2 of aorta intima-media, approximately 50% of subendothelial 125I-thrombin is bound to dermatan sulphate chains of proteoglycan in the extracellular matrix. The possibility is discussed that dermatan sulphate chains may function as thrombin-binding loci to control or augment thrombin activity in the ECM of the injured vascular wall in vivo.

Effects of the oral anticoagulant phenprocoumon on blood coagulation and thrombogenesis induced by rabbit aorta subendothelium exposed to flowing human blood: role of dose and shear rate.

We investigated the effect of oral anticoagulation on thrombogenesis induced by the subendothelium of rabbit aorta. Eighteen healthy volunteers underwent a 2-week treatment with the oral coumarin preparation phenprocoumon to a target international normalized ratio (INR) of 5. By using an ex-vivo perfusion chamber system, the interaction between flowing blood and exposed subendothelium was measured at low (50 sec-1) and high (650 sec-1) wall shear rates. The low shear rate simulated blood flow in venous vessels and the high shear rate simulated blood flow in arterial vessels. Deposition of fibrin, platelets, and platelet thrombi on subendothelium was quantified by morphometric and immunologic techniques. Fibrin deposition prevailed at the low shear rate (183 +/- 57 ng/mm2 vs 46 +/- 16 ng/mm2, low vs high shear rate; mean +/- SEM; p less than 0.01). In contrast, the interaction of platelets with subendothelium was more intense at high shear rates when compared with low shear rates, as indicated by higher platelet adhesion (44% +/- 2% vs 9% +/- 1% coverage of subendothelium with platelets, p less than 0.001) and platelet thrombus volumes (3.6 +/- 0.4 microns 3/microns 2 vs 1.3 +/- 0.3 microns 3/microns 2, p less than 0.001). Fibrin deposition on subendothelium was substantially reduced even at a low intensity of anticoagulation (reduction by 60% at INR 2.1 and by 75% at INR 3.7) and was abolished after high coumarin doses (INR 5.2). In contrast, a significant inhibition of platelet thrombus formation could be achieved only by high doses of phenprocoumon (INR 5.2). Our data indicate that relatively low doses of oral anticoagulants (INR between 2 and 3.5) substantially inhibit the fibrin formation on subendothelium prevailing at venous shear conditions, whereas a high intensity of anticoagulation (INR 4 to 5) is necessary to inhibit the formation of platelet-rich thrombi prevailing at arterial shear conditions.

Effects of low and high dose oral contraceptives on blood coagulation and thrombogenesis induced by vascular subendothelium exposed to flowing human blood

We investigated the effect of oral contraceptives with low and high estrogen concentration on blood coagulation and thrombogenesis, induced by vascular subendothelium of rabbit aorta exposed to flowing human blood. Twenty healthy women intending to take oral contraceptives were studied [1]before drug ingestion (control), and subsequently during the intake of oral contraceptives with [2]low estrogen content (20 μg ethinyl estradiol and 150 μg desogestrel per day) and [3] high estrogen content (50 μg ethinyl estradiol and 125 μg desogestrel per day). All experiments were performed between day 17 and 21 of the menstrual cycle and drug effects were studied during the third tablet cycle. Deposition of fibrin, platelets and platelet thrombi on vascular subendothelium was tested at a defined blood flow and wall shear rate (10 ml/min, 650 s .1) and was quantified by morphometrical techniques. Treatment with the low and high dose contraceptive increased the plasma levels of ethinyl estradiol (728 ± 139 and 1438 ± 212 vs. 0 fmol/ l [low and high dose vs. control], means ± SEM, P<0.001) and fibrinogen (2.3 ± 0.1 and 2.6 ± 0.1 vs. 2.0 ± 0.1 g/l, P<0.05); and decreased antithrombin III activity (95 ±3 and 92 ± 3 vs. 101 ± 3 %, P<0.05). Fibrin deposition on vascular subendothelium was enhanced by the high dose contraceptive only (47 ± 4 vs. 35 ±4 % coverage of the subendothelial surface with fibrin, high dose vs. control, P<0.05). The subendothelial deposition of platelets and platelet thrombi was not changed by contraceptive treatment. These results indicate that treatment with high dose contraceptives leads to an increase of fibrin-subendothelial interactions, whereas low dose contraceptives do not significantly alter the blood-subendothelium interactions observed in this ex vivo model of thrombogenesis.

Dose- and shear rate-dependent effects of heparin on thrombogenesis induced by rabbit aorta subendothelium exposed to flowing human blood.

The effect of heparin on thrombogenesis induced by the subendothelium of rabbit aorta was investigated in 24 healthy volunteers after intravenous injection of different doses (0, 1000, 2500, and 5000 IU). By using an ex vivo perfusion chamber system, the interaction between flowing blood and exposed subendothelium was measured at low (50 s-1), intermediate (650 s-1), and high (2600 s-1) wall shear rates. The low shear rate simulated blood flow in venous, the intermediate shear rate in arterial, and the high shear rate in small or stenosed arterial vessels. Deposition of fibrin, platelets, and platelet thrombi on vascular subendothelium (SE) was quantified by morphometrical and immunological techniques. Fibrin deposition prevailed at low shear rates and was only minimal at high shear rates (30 +/- 1% vs. 1 +/- 0.4% coverage of SE with fibrin, means +/- SEM, p less than 0.001). In contrast, the interaction of platelets with SE was more intense at high compared to low shear rates, as indicated by higher platelet adhesion (54 +/- 5% vs. 4 +/- 1% coverage of SE with platelets, p less than 0.001) and platelet thrombus volumes (4.8 +/- 1.3 vs. 0.5 +/- 0.1 microns 3/microns 2, p less than 0.001). Fibrin deposition on SE was inhibited by heparin in a dose-dependent manner and was abolished after high doses. In addition, high doses of heparin reduced the height and volume of platelet thrombi at low and intermediate wall shear rates, but no effect was found at the high shear rate. Our data show that heparin inhibits the formation of both fibrin and platelet thrombi on vascular subendothelium. The lack of effect of heparin on platelet thrombus formation at high shear rates indicates that thrombin modulates the growth rate and/or stability of platelet thrombi at low and intermediate shear rates, whereas additional factors may control platelet thrombus growth and stability at high shear conditions.

Evidence for the Presence of Tissue Factor Activity on Subendothelium

By a variety of methods, tissue factor activity was demonstrated in the subendothelium of rabbit aorta and human umbilical artery. In one method, everted segments of de-endothelialized vessels were mounted in an annular perfusion chamber and the subendothelial surface was exposed to nonanticoagulated human blood under controlled flow. Procoagulant activity was assessed by measuring fibrin deposition on subendothelium and fibrinopeptide A (FPA) levels in post chamber blood. Both fibrin deposition and FPA were decreased with rabbit vessel segments exposed (at a shear rate of 650 seconds-1) to blood from patients with factor VII deficiency and with umbilical artery segments (at shear rates of 90 to 180 seconds-1) that had been pretreated with a monoclonal antibody to human tissue factor. In a second method, everted umbilical artery segments were mounted on a stir bar and the subendothelial surface was exposed, with stirring, to plasma or purified coagulation factors. The capacity of the surface to clot plasma on addition of calcium was inhibited by the antibody to tissue factor. The surface also activated purified 3H-factor X in the presence of factor VIIa, but not in its absence, and this surface property was almost entirely eliminated by pretreating the vessel segments with antitissue factor. Tissue factor activity in subendothelium could play a role in both the arrest of bleeding and in promoting the formation of thrombi at sites of vascular injury.

Evidence for the presence of tissue factor activity on subendothelium

By a variety of methods, tissue factor activity was demonstrated in the subendothelium of rabbit aorta and human umbilical artery. In one method, everted segments of de-endothelialized vessels were mounted in an annular perfusion chamber and the subendothelial surface was exposed to nonanticoagulated human blood under controlled flow. Procoagulant activity was assessed by measuring fibrin deposition on subendothelium and fibrinopeptide A (FPA) levels in post chamber blood. Both fibrin deposition and FPA were decreased with rabbit vessel segments exposed (at a shear rate of 650 seconds-1) to blood from patients with factor VII deficiency and with umbilical artery segments (at shear rates of 90 to 180 seconds-1) that had been pretreated with a monoclonal antibody to human tissue factor. In a second method, everted umbilical artery segments were mounted on a stir bar and the subendothelial surface was exposed, with stirring, to plasma or purified coagulation factors. The capacity of the surface to clot plasma on addition of calcium was inhibited by the antibody to tissue factor. The surface also activated purified 3H-factor X in the presence of factor VIIa, but not in its absence, and this surface property was almost entirely eliminated by pretreating the vessel segments with antitissue factor. Tissue factor activity in subendothelium could play a role in both the arrest of bleeding and in promoting the formation of thrombi at sites of vascular injury.

Excessive Deposition of Fibrin, Platelets and Platelet Thrombi on Vascular Subendothelium During Contraceptive Drug Treatment

We investigated the effect of oral contraceptives on thrombogenesis induced by subendothelium of rabbit aorta (SE), exposed to flowing non-anticoagulated blood in an annular flow chamber. Six healthy women on sequential contraceptive drugs (0.05 mg aethinylestradiol/day, 0.125 mg desogestrel/day) were compared with 6 women without hormonal contraception and 6 men. On contraceptive drug treatment, blood values were significantly increased for fibrinogen (2.6 +/- 0.2 vs 1.9 +/- 0.1 g/l) and fibrinopeptide A (3.9 +/- 0.9 vs 0.9 +/- 0.1 ng/ml), whereas antithrombin III was decreased (81 +/- 4 vs 97 +/- 6%). Fibrin deposition on vascular subendothelium was more than four-fold increased when measured morphologically (63.4 +/- 2.5 vs 14.6 +/- 6.8% coverage of SE surface with fibrin) as well as immunologically (29.3 +/- 2.2 vs 4.5 +/- 1.9 micrograms fibrin/cm2 of SE). Thrombus volumes were more than two-fold increased in women with contraceptives (9.0 +/- 1.4 vs 3.7 +/- 1.0 micron 3/micron 2). Our study shows that during contraceptive drug treatment the exposure of flowing blood to vascular subendothelium leads to excessive deposition of fibrin and platelet thrombi. Measurement of blood interactions with subendothelium might be of predictive value in hypercoagulable states such as contraceptive treatment.

Factor VIII/von Willebrand factor in subendothelium mediates platelet adhesion

The present studies were undertaken to determine whether factor VIII/von Willebrand factor (vWF) present in the vessel wall (in addition to that in plasma) may mediate the attachment of platelets to subendothelium. Subendothelium from everted rabbit aorta was exposed to human citrated blood flowing through an annular perfusion chamber at 40 mL/min (wall shear rate of 2,600 s-1 for five minutes). The vessel segments were incubated at 37 degrees C for one hour with various dilutions of either goat-anti-rabbit factor VIII/vWF serum or an IgG fraction prepared from the serum. Control segments were incubated with serum or IgG from a nonimmunized goat. Values of platelet contact (C), platelet adhesion (C + S), and thrombus formation (T) on the subendothelium were evaluated by a morphometric technique. Compared with vessels incubated with fractions prepared from a normal goat, a significant decrease in platelet adhesion (C + S), ranging from 45% to 65%, was observed on vessels incubated with various dilutions (1:5 to 1:50) of either serum or IgG fractions of goat-anti-rabbit factor VIII/vWF. A similar decrease in platelet adhesion was observed with vessels incubated with an F(ab')2 fragment against rabbit factor VIII/vWF prepared in the goat. When goat-anti-rabbit factor VIII/vWF IgG was added to rabbit blood (1:75 dilution), platelet adhesion was reduced to the same extent (65%) on normal rabbit vessels and on vessels pre-incubated with goat-anti-rabbit factor VIII/vWF. Immunofluorescence studies revealed the presence of rabbit factor VIII/vWF in the subendothelium of rabbit aorta and the continued binding of the goat-anti-factor VIII/vWF antibodies on subendothelium during the perfusion studies. No uptake of human factor VIII/vWF on the rabbit subendothelium was observed by this immunologic technique; human factor VIII/vWF was found to be entirely associated with the attached human platelets. Thus, factor VIII/vWF in the vessel wall may mediate platelet attachment to subendothelium in a manner similar to that of plasma factor VIII/vWF.

Factor VIII/von Willebrand Factor in Subendothelium Mediates Platelet Adhesion

Abstract The present studies were undertaken to determine whether factor VIII/von Willebrand factor (vWF) present in the vessel wall (in addition to that in plasma) may mediate the attachment of platelets to subendothelium. Subendothelium from everted rabbit aorta was exposed to human citrated blood flowing through an annular perfusion chamber at 40 mL/min (wall shear rate of 2,600 s-1 for five minutes). The vessel segments were incubated at 37 degrees C for one hour with various dilutions of either goat-anti-rabbit factor VIII/vWF serum or an IgG fraction prepared from the serum. Control segments were incubated with serum or IgG from a nonimmunized goat. Values of platelet contact (C), platelet adhesion (C + S), and thrombus formation (T) on the subendothelium were evaluated by a morphometric technique. Compared with vessels incubated with fractions prepared from a normal goat, a significant decrease in platelet adhesion (C + S), ranging from 45% to 65%, was observed on vessels incubated with various dilutions (1:5 to 1:50) of either serum or IgG fractions of goat-anti-rabbit factor VIII/vWF. A similar decrease in platelet adhesion was observed with vessels incubated with an F(ab')2 fragment against rabbit factor VIII/vWF prepared in the goat. When goat-anti-rabbit factor VIII/vWF IgG was added to rabbit blood (1:75 dilution), platelet adhesion was reduced to the same extent (65%) on normal rabbit vessels and on vessels pre-incubated with goat-anti-rabbit factor VIII/vWF. Immunofluorescence studies revealed the presence of rabbit factor VIII/vWF in the subendothelium of rabbit aorta and the continued binding of the goat-anti-factor VIII/vWF antibodies on subendothelium during the perfusion studies. No uptake of human factor VIII/vWF on the rabbit subendothelium was observed by this immunologic technique; human factor VIII/vWF was found to be entirely associated with the attached human platelets. Thus, factor VIII/vWF in the vessel wall may mediate platelet attachment to subendothelium in a manner similar to that of plasma factor VIII/vWF.

Platelet interaction with rabbit subendothelium in von Willebrand's disease: altered thrombus formation distinct from defective platelet adhesion.

Blood interaction with the subendothelium of rabbit aorta was investigated in an annular perfusion chamber using patients with von Willebrand's disease, hemophilia, and afibrinogenemia. The vessels were exposed to nonanticoagulated blood for a range of flow conditions (wall shear rates of 650-3,300 s-1) and exposure times (1.5-10 min). The resultant platelet and fibrin interaction was quantified by the use of several morphometric techniques, one of which was developed to measure more precisely the dimensions (height and volume) of platelet thrombi attached to the subendothelium. A major finding was that under flow conditions in which little or no defect in platelet adhesion was observed in von Willebrand's disease, platelet thrombus height and volume in this disorder were significantly reduced as compared with normal controls or patients with hemophilia. Thus, Factor VIII/von Willebrand factor (VIII/VWF) may mediate not only the adhesion of platelets to subendothelium but also platelet-platelet attachments necessary for normal thrombus development. The level of Factor VIII:coagulant activity (VIII: C) was also observed to influence the resultant thrombus height and volume deposited on subendothelium, presumably through the generation of thrombin or some other procoagulant factor preceding fibrin formation, since normal values of thrombus dimensions were always observed in a patient with a fibrinogen deficiency. The influence of VIII:C became greater as shear rate was reduced, whereas as shear rate was increased, VIII/VWF was more dominant in determining the resultant platelet deposition on subendothelium. Thus, the deficiencies of VIII:C and VIII/VWF in hemophilia and von Willebrand's disease can lead to various abnormalities in platelet and fibrin association with subendothelium. The importance of a particular deficiency will depend strongly on the local blood flow conditions.

Monoclonal antibody to human platelet glycoprotein I. II. Effects on human platelet function.

The effect on platelet function of a monoclonal platelet antibody to platelet membrane glycoprotein I was tested. This antibody, AN51, inhibited ristocetin or bovine factor VIII-induced aggregation but did not modify ADP, collagen type I or type III, thrombin or arachidonic acid induced aggregations. Furthermore, the adhesion-aggregation of platelets induced by microfibrils was also inhibited by the antibody. Platelet adhesion to rabbit aorta subendothelium was impaired by the antibody. The persistent adhesion of platelets to collagenase-treated subendothelium was also inhibited. These findings strongly suggested that platelet membrane glycoprotein I could interact with a non-collagenic microfibrillar component of subendothelium. The binding of factor VIII/von Willebrand factor to platelet membrane in the presence of ristocetin was decreased in the binding site for factor VIII/von Willebrand factor to allow platelet adhesion to subendothelium.

Effects of acetylsalicylic acid, sulfinpyrazone and dipyridamole on platelet adhesion and aggregation in flowing native and anticoagulated blood.

The interaction of rabbit platelets with subendothelium of rabbit aorta was investigated under controlled blood flow conditions. Platelet adhesion and thrombus formation were compared after perfusion of native blood and of blood anticoagulated with citrate, heparin or heparin plus citrate. 15 mM citrate in plasma caused significant reduction of aggregation, thrombus volume and thrombus height; adhesion was concomitantly increased. Heparin (500 U/kg) had no significant effect on adhesion and thrombus dimensions. Treatment of rabbits with acetylsalicylic acid or sulfinpyrazone caused a significant reduction of thrombus volume and thrombus height in the presence of citrate. However, no significant effects were observed in native or heparinized blood. It is concluded that low citrate concentrations: (1) inhibit thrombus growth; (2) enhance thrombus breakdown; (3) therefore increase adhesion, and (4) strongly enhance a possible inhibitory effect of acetylsalicylic acid and sulfinpyrazone on thrombus growth.

Adhesion and aggregation of human platelets to rabbit subendothelium. A new approach for investigation: specific antibodies.

An SgG antibody occurring in a recently transfused thrombasthenic patient inhibited all the ADP-mediated aggregations and platelet-platelet interaction (thrombus formation) on rabbit aorta subendothelium; another IgG antibody occurring in a multitransfused Bernard-Soulier patient inhibited ristocetin and bovine factor VIII mediated aggregation and platelet-subendothelium interaction.

Defective Platelet Adhesion and Aggregation on Subendothelium Exposed in Vivo or in Vitro to Flowing Blood of Fawn-Hooded Rats with Storage Pool Disease

SummaryCitrated rat blood was exposed to either subendothelium or the fibrillar collagen of enzymatically modified subendothelium of rabbit aorta in a perfusion system under laminar blood flow conditions at a wall shear rate of 830 s−1. The resulting platelet surface interaction was estimated by a morphometric method.With blood of fawn-hooded (FH) rats, which suffer from hereditary platelet “storage pool disease”, platelet spreading was slower on both exposed surfaces and resulted in a lower rate of surface coverage with platelets on subendothelium if compared with controls.The rate of adhesion of FH-platelets to the fibrillar collagen, however, was slightly higher as compared to controls despite reduced platelet spreading. This was probably due to the absence of platelet thrombus formation observed with FH-rat blood, whereas massive platelet thrombus formation took place in the controls. It is suggested that platelets of controls which arrive near the surface are preferentially incorporated into the rapidly forming platelet thrombi rather than reaching the surface, and hence do not increase surface-coverage with adhering platelets.The defective platelet adhesion and aggregation in the FH-rat was also apparent after desendothelialization of the aorta in vivo, although to a lesser extent, probably due to the extremely low thrombogenicity of rat aorta subendothelium.

Physiological Experiments in Haemostasis and Thrombosis

Summary.An in vitro perfusion system has been developed which allows morphometric measurement of platelet adhesion and surface‐induced aggregation after exposure of subendothelium and other surfaces to flowing blood. Recent results are briefly reviewed and compared with results obtained using other methods.A number of rheologic and blood‐borne factors including flow velocity, vessel geometry, presence of red blood cells and divalent cations, affect both platelet adhesion and surface‐induced aggregation.Diminished adhesion of platelets to subendothelium of rabbit aorta but normal surface‐induced aggregation was found after exposure to blood of patients with von Willebrand's disease and the Bernard‐Soulier syndrome at blood flow rates similar to those found in arteries. In contrast, surface‐induced aggregation was defective in patients with thrombasthenia and in an inbred strain of rats with a haemorrhagic diathesis, similar to the platelet ‘storage pool disease’in patients. Similarly, normal adhesion and strikingly diminished aggregation were found with blood of rabbits after aspirin ingestion. These findings may explain the defective haemostasis in the patients and animals, and suggest a new way to study platelet function in a physiological system.