We have developed a model of acute Dacron graft thrombosis in baboons in order to assess platelet alterations secondary to arterial thrombus formation. In this model, thrombus formation was initiated by Dacron vascular grafts inserted as extension segments into chronic arteriovenous Silastic shunts. Following platelet labeling with 111In-oxine, platelet deposition was measured for 1 hour following blood contact under arterial flow conditions using a scintillation camera. Graft platelet activity rapidly increased 40- to 50-fold, plateauing by 1 hour. All grafts produced equivalent reductions in circulating platelet count and blood 111In-platelet radioactivity, demonstrating that the labeled cells were functionally equivalent to the total platelet population. After graft placement, the remaining platelets survived normally. Acute platelet deposition was equivalent on grafts placed 1, 24, 48, and 72 hours following injection of the labeled cells, indicating that a variable delay between platelet labeling and graft imaging was without detectable consequence. Platelet destruction by the graft produced a tenfold increase in plasma levels of platelet factor 4 (PF4) and beta-thromboglobulin (beta TG) but did not modify either the alpha-granule (PF4, beta TG) or dense granule (ADP, ATP) contents of circulating platelets.

Abstract-Platelet adhesion in arterial blood flow is mainly supported by the platelet receptor glycoprotein (GP) Ib, which interacts with von Willebrand factor (vWF) that is bound to collagen at the site of vessel wall injury. Antibody 6B4 is a monoclonal antibody (MoAb) raised against purified human GPIb. MoAb 6B4 inhibits both ristocetin-and botrocetin-induced, vWF-dependent human platelet agglutination. MoAb 6B4 furthermore blocks shear-induced adhesion of human platelets to collagen I. We studied the antithrombotic effect of this inhibitory murine MoAb 6B4 in a baboon model of arterial thrombosis. When injected into baboons, intact IgG and its F(abЈ) 2 fragments caused almost immediate thrombocytopenia, whereas injection of the Fab fragments alone did not. Fab fragments were subsequently used to investigate their in vivo effect on platelet deposition onto a thrombogenic device, consisting of collagen-rich, glutaraldehyde-fixed bovine pericardium (0.6 cm 2 ), at a wall shear rate ranging from 700 to 1000 s Ϫ1 . Baboons were either pretreated with Fabs to study the effect of inhibition on platelet adhesion or treated 6 minutes after placement of the thrombogenic device to investigate the effect on interplatelet cohesion. Pretreatment of the animals with bolus doses ranging from 80 to 640 g/kg Fab fragments significantly reduced 111 In-labeled platelet deposition onto the collagen surface by Ϸ43% to 65%. Only the highest dose caused a significant prolongation (doubling) of the bleeding time. Ex vivo ristocetin-induced platelet agglutination was equally reduced. Treatment with a bolus of 110 g/kg Fab fragments after a thrombus was allowed to form for 6 minutes had no effect on further platelet deposition. We therefore conclude that Fab fragments or derivatives of inhibitory anti-GPIb antibodies may be useful compounds to prevent thrombosis.

The interaction between collagen, von Willebrand factor (VWF), and glycoprotein Ib is the first step in hemostasis and thrombosis especially under high shear conditions. We studied the inhibition of the VWF-collagen interaction by using an antihuman VWF monoclonal antibody 82D6A3 to prevent arterial thrombosis in baboons to develop a new kind of antithrombotic strategy and determine for the first time experimental in vivo data concerning the importance of the collagen-VWF interaction. We used a modified Folts model to study the antithrombotic efficacy of 82D6A3, where cyclic flow reductions (CFRs) were measured in the femoral artery. Administering a dose of 100, 300, and 600 g/kg resulted in a 58.3%, 100%, and 100% reduction in the CFRs, respectively. When 100 g/kg 82D6A3 was infused into the baboons, 80% of VWF-A3 domain was occupied, corresponding to 30% to 36% ex vivo inhibition of VWF binding to collagen, with no prolongation of the bleeding time. The bleeding time was also not significantly prolonged when the CFRs were abolished at doses of 300 g/kg and 600 g/kg. At these doses 100% of VWF was occupied by the antibody and 100% ex vivo inhibition of the VWF-collagen binding was observed. 82D6A3 has a high affinity for VWF; after 48 hours still 68% VWF (300g/kg) was occupied with a pharmacologic effect up to 5 hours after administration (80%-100% occupancy). In conclusion, these results clearly indicate that the VWFcollagen interaction is important in vivo in thrombosis under high shear conditions and thus might be a new target for preventing arterial thrombosis. (Blood. 2002;99:3623-3628)

Abstract-The antithrombotic efficacy of the monoclonal antibodies 6B4-Fab and MA-16N7C2 against platelet glycoprotein (GP) Ib and GP IIb/IIIa, respectively, on acute platelet-mediated thrombosis was evaluated in a baboon model of femoral artery stenosis, which is a modification of the original Folts model: platelet thrombi form on the injured stenosed artery, producing cyclic flow reductions (CFRs). A dose of 0.6 mg/kg 6B4-Fab significantly reduced the CFRs by 59Ϯ15%, whereas 2 mg/kg 6B4-Fab completely abolished the CFRs without prolongation of the bleeding time. MA-16N7C2 inhibited CFRs by 43Ϯ8% at a dose of 0.1 mg/kg and abolished the CFRs at a dose of 0.3 mg/kg but with a significant prolongation of the bleeding time. Finally, the combination of 0.6 mg/kg 6B4-Fab and 0.1 mg/kg MA-16N7C2 fully prevented the CFRs without prolongation of the bleeding time. The present study demonstrates that the inhibition of platelet GP Ib function by 6B4-Fab is a powerful intervention to prevent platelet thrombus formation in injured arteries without prolongation of the bleeding time; the latter is in contrast to the result after the inhibition of GP IIb/IIIa. Moreover, we demonstrate that combining a GP Ib blocker with a GP IIb/IIIa blocker can achieve a strong antithrombotic effect without increasing the bleeding time. This provides new information that will be beneficial in designing clinical therapeutic approaches. Key Words: platelet glycoprotein Ib Ⅲ platelet glycoprotein IIb/IIIa Ⅲ cyclic flow reductions Ⅲ antithrombotic agents Ⅲ bleeding time P latelets adhere to the subendothelium of damaged blood vessels through the collagen-von Willebrand factor (vWF)-platelet glycoprotein (GP) Ib axis. vWF forms the bridge between platelets and collagen in the vessel wall (especially under high-shear conditions) and in stenosed arteries and microvessels, where it initiates the formation of platelet aggregates. When vWF binds to GP Ib, platelets are slowed down, allowing direct platelet-collagen receptor interactions via, for example, integrin ␣ 2 ␤ 1 and glycoprotein VI, which activate platelets, finally resulting in a conformational change in the GP IIb/IIIa receptor to enable binding to fibrinogen and vWF, leading to the formation of platelet aggregates. 1,2 After extensive vessel injury or rupture of atherosclerotic plaques with subsequent exposure of thrombogenic surfaces, platelet aggregation can progress to result in complete thrombotic occlusion of the injured vessel. 3,4 Various clinical studies and studies in experimental animals have clearly shown that inhibition of platelet aggregation, through prevention of the binding of the adhesive proteins to GP IIb/IIIa, is an effective approach to prevent thrombosis. [5][6][7][8] Unfortunately, this approach increases the risk of bleeding, especially at the doses that are effective in preventing thrombotic episodes. 8 Blocking GP Ib 9 -11 or vWF 12 results in an inability of the platelets to attach to the exposed subendothelium. Therefore, the GP Ib-vWF axis is an attractive targe...

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The purpose of this study was to determine the anticoagulant and antithrombotic potential of hirudin during hemodialysis by comparing the efficacy of dialysis with heparin to that of dialysis with recombinant hirudin (r-hirudin). Eleven patients with chronic renal failure and on maintenance hemodialysis were included in this open cross-over study. Conventional doses of heparin were administered during the first dialysis of the study. Two days later r-hirudin, at a dose of 0.15 mg/kg, was given as a bolus at the start of the second dialysis. The mean decreases in plasma levels of urea, uric acid and creatinine were approximately 50% after dialysis with both anticoagulants. Dialysis was therefore equally effective. However, effective dialysis with r-hirudin was achieved with a shorter activated partial thromboplastin time (APTT; range 65 to 103 seconds) compared to that with heparin (> 120 seconds), thereby decreasing the risk of bleeding. Markedly less 111In-labeled platelets accumulated at the inlet of the artificial kidney when r-hirudin was used, suggesting a smaller loss of hollow fiber volume. The results indicate that hirudin may be a suitable alternative anticoagulant for use during hemodialysis and it thus warrants further investigation.

Three groups of subjects (6 subj in each group) underwent the following precedures: group A was given a 20-min head-up tilt at 21 degrees C followed by 4 h of exercise at 33.9 degrees C DB, 32.2 degrees C WB, and a repetition of tilting after exercise in heat; group B underwent the same procedure at 21 degrees C; group C was tilted at 21 degrees C, rested in heat for 4 h and was retilted in heat. The above procedures were repeated for 8 days, and on the last day groups B and C underwent the same treatment as group A. Group A showed the usual decreases in heart rate and rectal temperature and an increase in sweat rate on acclimation. This corresponded to marked improvements in heat-orthostatism. While five subjects in group A fainted during post-exposure tilting on the first exposure, none fainted on the last day. Resting in heat (group C) did not cause any acclimation to work in heat. This corresponded to poor heat-orthostatism after the work-heat procedure when five subjects fainted. Mild training at 21 degrees C (group B) resulted in minor improvements to work in heat as evident by some improvements in heart rate responses after the 1st and 2nd h of exposure. This corresponded to better heat-orthostatism and fewer men fainting than in group C. The results indicated that heat-orthostatism improves on acclimation to the same extent as exercise heart rate and rectal temperature.

SummaryPlatelets were isolated from blood of baboons and treated with neuraminidase to remove platelet membrane sialic acid, a process which artificially ages the platelets. The platelets were then labelled with 111In and their mean life span, in vivo distribution and sites of Sequestration were measured. The effect of removal of sialic acid on the attachment of immunoglobulin to platelets were investigated and related to the Sequestration of the platelets by the spleen, liver, and bone marrow. Removal of sialic acid by neuraminidase did not affect the aggregation of platelets by agonists in vitro, nor their sites of Sequestration. The removal of 0.51 (median, range 0.01 to 2.10) nmol sialic acid/108 platelets shortened their life span by 75 h (median, range 0 to 132) h (n = 19, p <0.001), and there was an exponential correlation between the shortening of the mean platelet life span and the amount of sialic acid removed. The increase in platelet-associated IgG was 0.112 (median, range 0.007 to 0.309) fg/platelet (n = 25, p <0.001) after 0.79 (median, range 0.00 to 6.70) nmol sialic acid/108 platelets was removed (p <0.001). There was an exponential correlation between the shortening of mean platelet life span after the removal of sialic acid and the increase in platelet-associated IgG. The results suggest that platelet membrane sialic acid influences ageing of circulating platelets, and that the loss of sialic acid may have exposed a senescent cell antigen that binds IgG on the platelet membrane. The antibody-antigen complex may then provide a signal to the macrophages that the platelet is old, and can be phagocytosed and destroyed.