Arterial thrombosis is considered to arise from the interaction of tissue factor (TF) in the vascular wall with platelets and coagulation factors in circulating blood. According to this paradigm, coagulation is initiated after a vessel is damaged and blood is exposed to vessel-wall TF. We have examined thrombus formation on pig arterial media (which contains no stainable TF) and on collagen-coated glass slides (which are devoid of TF) exposed to f lowing native human blood. In both systems the thrombi that formed during a 5-min perfusion stained intensely for TF, much of which was not associated with cells. Antibodies against TF caused Ϸ70% reduction in the amount of thrombus formed on the pig arterial media and also reduced thrombi on the collagencoated glass slides. TF deposited on the slides was active, as there was abundant fibrin in the thrombi. Factor VII ai , a potent inhibitor of TF, essentially abolished fibrin production and markedly reduced the mass of the thrombi. Immunoelectron microscopy revealed TF-positive membrane vesicles that we frequently observed in large clusters near the surface of platelets. TF, measured by factor X a formation, was extracted from whole blood and plasma of healthy subjects. By using immunostaining, TF-containing neutrophils and monocytes were identified in peripheral blood; our data raise the possibility that leukocytes are the main source of blood TF. We suggest that blood-borne TF is inherently thrombogenic and may be involved in thrombus propagation at the site of vascular injury.Tissue factor (TF) present in the arterial wall has been considered to be responsible for the initiation of the coagulation cascade and thrombus formation (1). The role of vascular TF in acute thrombosis and atherosclerosis has been proposed based on our previous studies (2-5). To investigate the role of circulating TF in thrombogenesis, we have used a system in which pig aortic media or collagen-coated slides were mounted in a laminar flow chamber and perfused with native human blood. We noted that when stained either with derivatized factor VII a (FVII a ) or with specific anti-TF antibodies, the thrombi contained large amounts of TF staining, whereas the media and collagen-coated slides were essentially negative. Thus, we surmised that the TF came from the blood; accordingly, we examined whole blood and plasma for TF activity that we have extracted and assayed. We conclude that there is circulating, potentially active TF in normal subjects. We present evidence that this pool is thrombogenic in model flow systems. We also present evidence suggesting the TF comes from leukocytes and hypothesize that the cell-surface TF is completely encrypted (6-8) but becomes available during thrombosis. METHODSReagents. Human recombinant FVII a was a gift from NovoNordisk, Copenhagen. Factor X was purified from human plasma (9). Affigel-15 was purchased from Bio-Rad. The phospholipids used for relipidation of TF consisted of 30% 1,2-dioleoyl-sn-glycero-3-phosphatidylserine and 70% 1,2-dioleoyl-sn-gl...

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Fabry disease results from deficient alpha-galactosidase A (alpha-Gal A) activity and the pathologic accumulation of the globotriaosylceramide (GL-3) and related glycosphingolipids, primarily in vascular endothelial lysosomes. Treatment is currently palliative, and affected patients generally die in their 40s or 50s. Preclinical studies of recombinant human alpha-Gal A (r-halphaGalA) infusions in knockout mice demonstrated reduction of GL-3 in tissues and plasma, providing rationale for a phase 1/2 clinical trial. Here, we report a single-center, open-label, dose-ranging study of r-halphaGalA treatment in 15 patients, each of whom received five infusions at one of five dose regimens. Intravenously administered r-halphaGalA was cleared from the circulation in a dose-dependent manner, via both saturable and non-saturable pathways. Rapid and marked reductions in plasma and tissue GL-3 were observed biochemically, histologically, and/or ultrastructurally. Clearance of plasma GL-3 was dose-dependent. In patients with pre- and posttreatment biopsies, mean GL-3 content decreased 84% in liver (n=13), was markedly reduced in kidney in four of five patients, and after five doses was modestly lowered in the endomyocardium of four of seven patients. GL-3 deposits were cleared to near normal or were markedly reduced in the vascular endothelium of liver, skin, heart, and kidney, on the basis of light- and electron-microscopic evaluation. In addition, patients reported less pain, increased ability to sweat, and improved quality-of-life measures. Infusions were well tolerated; four patients experienced mild-to-moderate reactions, suggestive of hypersensitivity, that were managed conservatively. Of 15 patients, 8 (53%) developed IgG antibodies to r-halphaGalA; however, the antibodies were not neutralizing, as indicated by unchanged pharmacokinetic values for infusions 1 and 5. This study provides the basis for a phase 3 trial of enzyme-replacement therapy for Fabry disease.

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