Bonnie Ross

Pretreatment of Rabbits With Either Hirudin, Ancrod, or Warfarin Significantly Reduces the Immediate Uptake of Fibrinogen and Platelets by the Deendothelialized Aorta Wall After Balloon-Catheter Injury In Vivo

Fibrinogen and platelets rapidly saturate the exposed subendothelium of a freshly deendothelialized aorta in vivo. As thrombin generated within the site of injury is largely responsible for fibrin(ogen) deposition, we questioned whether various anticoagulant treatments would inhibit uptake of both fibrinogen and platelets in vivo. Rabbits were anticoagulated by pretreatment with either Warfarin, Ancrod, or recombinant hirudin. Each anesthetized, anticoagulated (or saline-injected control) rabbit was injected i.v. with rabbit 51Cr-platelets and 125I-fibrinogen before a balloon-catheter deendothelializing (or sham) injury of the thoracic aorta. At 10 minutes after injury, the rabbit was exsanguinated and the aorta excised. Platelet adsorption by the deendothelialized aorta surface was substantially reduced in anticoagulated rabbits (controls, 2.2x10(5)/mm2; Warfarin-treated, 1.2x10(5)/mm2; Ancrod-treated, 5.3x10(4)/mm2; r-hirudin-treated [5 mg/kg], 5.3x10(4)/mm2), and a significant reduction of fibrinogen associated with the platelet layer (from 5.3 to 1 to 2 pmol/cm2) and within the underlying intima-media layer (from 16.9 to 5 to 6 pmol/cm2) was observed in the r-hirudin-and Warfarin-treated rabbits. The pattern of aorta-deposited 51Cr-platelets and 125I-fibrin in the anticoagulated rabbits corresponded well with an assessment by transmission electron microscopy of aortic tissue samples. We conclude that approximately 70% of fibrinogen uptake is thrombin dependent and that approximately 80% of platelet adsorption depends on codeposited fibrin(ogen) during the 10-minute interval after balloon injury. Pretreatment with an agent that interferes with either thrombin or fibrin production will inhibit the immediate interaction of fibrinogen and platelets with the freshly exposed subendothelium.

Plasminogen II accumulates five times faster than plasminogen I at the site of a balloon de-endothelializing injury in vivo to the rabbit aorta: Comparison with other hemostatic proteins

In the rabbit blood stream, plasminogen circulates as two glycoforms, plasminogen I (PLG-I) and plasminogen II (PLG-II), in a molar ratio of 1:2.2. To compare their relative behaviors toward a site of vascular injury, radiolabeled samples of PLG-I and PLG-II were coinjected intravenously into NZW rabbits before inducing a de-endothelializing (balloon catheter) injury to the thoracic aorta. At various times (5 to 60 minutes) after injury, each rabbit was anesthetized and exsanguinated, the aorta was excised, and the radioactivity per centimeters squared of aortic intima-media (IM) was measured relative to that of blood at exsanguination. The uptake of iodine 125-labeled PLG-I and iodine 131-labeled PLG-II showed that the IM was essentially saturated by both glycoforms by 30 to 40 minutes after injury. Extrapolation of the flux rates to 1 minute after injury indicated that the uptake of PLG-II (2.4 pmol/min/cm2) exceeded PLG-I (0.5 pmol/min/cm2) almost five-fold. This result is consistent with an earlier report (Metabolism 1994;43:1430-7) that PLG-II is released by the liver and catabolized in vivo approximately five times faster than PLG-I. By molar comparison, the flux of total plasminogen (ie, PLG-I plus PLG-II) into the injured aorta wall in vivo was 2.4 times greater than that for prothrombin. Assuming both zymogens are converted to their respective proteases within the wound site, then approximately 2 to 3 molecules of plasmin are released for each molecule of thrombin in vivo. The possible significance of this plasmin:thrombin ratio is discussed in respect to the turnover of fibrin(ogen) within the site of vascular injury.

Platelet and fibrinogen turnover at the exposed subendothelium measured over 1 year after a balloon catheter de-endothelializing injury to the rabbit aorta:: Thrombotic eruption at the late re-endothelialization stage

Balloon catheter de-endothelialization of the rabbit aorta in vivo causes a rapid release of thrombin and a consequent hemostatic response at the surface of the exposed subendothelium. Previously, we have compared the net fluxes of several hemostatic proteins from plasma into the exposed aorta subendothelium for up to 600 days after injury. We now report the turnover of platelets, compared to fibrinogen, at the de-endothelialized aorta for up to 390 days after injury. Anesthetized NZW rabbits received either a de-endothelializing or a sham injury (controls) to their aortas. At a predetermined time (either 10 min before or up to 390 days after injury), each rabbit was infused with known quantities of rabbit (51)Cr-platelets and rabbit (125)I-fibrinogen; the radiolabels were allowed to circulate for 10 min before the rabbit was rapidly exsanguinated. Radioactivity measurements and tissue analysis revealed that at 10 min after balloon injury, approximately 165,000 platelets/mm(2) were associated with the aorta surface, and platelet turnover was 840/min/mm(2). Turnover had decreased to <200/min/mm(2) at 10-21 days but, from 65 to 390 days, had increased to approximately 1500/min/mm(2). In comparison, approximately 17 pmol of fibrinogen/cm(2) saturated the ballooned surface by 10 min after injury. Fibrinogen turnover at the aorta surface at 10 min after injury amounted to 0.2 pmol/min/cm(2), increasing to 0.7 at 10 days but decreasing to 0.25 at 21 days. Between 65 and 390 days, fibrinogen turnover increased slowly to 1.3 pmol/min/cm(2). Fibrinogen turnover at the surface of the aorta paralleled that within the intima-media over 390 days. Platelet and fibrin(ogen) deposits within the aorta wall increased over the 21-390 days interval as shown by immunostaining. The results are consistent with the re-endothelializing aorta tending to support thrombosis and ulceration in the late healing stage.

Turnover and fate of fibrinogen and platelets at the rabbit aorta wall immediately after a balloon de-endothelializing injury in vivo

A de-endothelializing injury to the artery wall in vivo results in a rapid procoagulant response at the surface of the exposed subendothelium. Activated tissue factor (TF)-bearing cells and hemostasis factors located at the site of injury respond by producing thrombin, and within minutes the principal thrombus-forming, blood-borne components (platelets, fibrinogen) accumulate at the site. To compare their behaviors, the rates of uptake and turnover of rabbit (51)Cr-platelets and rabbit (125)I-fibrinogen were quantified simultaneously during the initial 100-min interval after a balloon catheter injury to the rabbit aorta in vivo. Platelets ( approximately 70,000/mm(2)) and fibrin(ogen) ( approximately 2.8 pmol/cm(2)) saturated the ballooned aorta surface within five minutes after injury. Whereas the adherent platelet and fibrinogen concentrations remained steady at the aorta surface, fibrin(ogen)-related products continued to accumulate slowly in the tunica media (TM) for at least 100 minutes. A relatively small proportion (3.7%/min) of adhered platelets turned over at the ballooned aorta surface at 10 minutes, decreasing to 1.2%/min at 100 minutes. By contrast, a larger proportion of fibrin(ogen) ( approximately 20%/min) was turned over within the platelet layer at 10 minutes, decreasing to 6%/min at 100 minutes. As verified by immunostaining aorta sections and by protein analysis of TM extracts, the uptakes of platelets and fibrinogen at the site of injury contributed to an accumulation of products of platelet releasate and fibrin(ogen) degradation (FDPs) within the TM. These observations improve our understanding of the hemostatic processes and subsequent events that occur after an arterial injury in vivo.