Chesebro, James H.

SummaryReversibility in vivo of acute platelet thrombosis in response to specific anticoagulants is analyzed with thrombi that develop in segments (1 cm) of porcine carotid arteries externally crushed with a hemostat. Most thrombi fill the lumens of the injured segments (ca. 1 cm × 3 mm, 1 × w) within 30 min and comprise masses of platelets interpenetrated with neutrophil-lined seepage channels of blood. Continuous quantitative assay of thrombus mass is provided by a gamma detector placed over the injured segments to collect counts from 111In-labeled platelets. Thrombi established 30 min after injury, otherwise stable for 6 h, clear during 30-60 min of continuous infusion of either hirudin, tick anticoagulant or activated porcine protein C, or intermittent activation of endogenous protein C with a latent thrombin reagent. Anticoagulant dose-dependence of thrombus clearance is established for hirudin between 0.01 and 1.0 mg/kg/min. Thrombi become progressively refractory to hirudin between 0.5 and 6 h after injury. Neither heparin nor low-molecular-weight heparin in full (clinical) anticoagulant doses yield significant dethrombosis. It is concluded that, within time limits, controlled thrombin generation in platelet thrombi maintains platelet cohesion without catalyzing irreversible platelet aggregation or clotting of fibrinogen.

The study comprised 170 consecutive patients (pts) seen at Mayo Clinic between 1962 and 1970 who underwent mitral valve replacement with Starr-Edwards prosthesis (model 6000 in 35% of pts, model 6120 in 65% of pts) because of significant chronic mitral incompetence (functional Class III-IV). All patients were on oral anticoagulants and were followed until January 1980 with a followup of 10 to 19 years (yrs) (median 15 yrs). We analyzed (1) the cumulative incidence of systemic emboli, their location and whether or not they left residual deficit; and (2) possible risk factors that might have been predictive, including in the analysis: age, sex, functional class, presence or absence of atrial fibrillation, degree of cardiomegaly, type of prosthesis and degree of anticoagulation.At 12 yrs of followup, 48% of pts had at least one episode of emboli (among these pts, 12% had two episodes); the rate of emboli was maximal within 1 yr postoperatively but persisted over the total followup at a rate of about 3% per yr. Of all emboli, 94% occurred in the cerebral circulation and 66% left neurological deficit. Regarding predictive factors, the incidence of emboli was slightly higher in pts with the prosthesis model 6000 (p<0.05) and in pts considered to be poorly anticoagulated (p<0.05).This unique long-term followup study on pts with mitral Starr-Edwards prosthesis reveals that systemic emboli is a persistent and significant problem. Preliminary data indicates that the addition of a platelet inhibitor to the oral anticoagulant may be beneficial.

The incidence of reinfarction, death and systemic thromboembolism is greatest in the first 2 months after acute myocardial infarction and decreases with time after infarction. Reasons for this are unknown, but increased platelet vascular wall interaction may be a contributing factor. Thirteen patients, mean age 53 years, had a platelet survival determination (platelet half-life calculated by least square analysis) within 1 month (mo) after acute, uncomplicated myocardial infarction (10 transmural, 3 subendocardial); a platelet survival study was repeated 3-8 mo (average 4 mo) later. All patients were on oral anticoagulants and during the time of observation none of the patients had any clinical thromboembolic events.Ten of the 13 patients had a shortened (<92 hours) platelet survival half-life of 75 hours (avg) within 1 mo after myocardial infarction which increased significantly (P<.01) to 94 hours (avg) four mo (avg) later. Ten control patients with angiographically proven coronary artery disease, stable angina pectoris, and a shortened platelet survival of 85 hours (avg) had a repeat platelet survival 6 mo later that was not significantly changed (mean variability 5%).The shortened platelet survival that improves with time after myocardial infarction is compatible with the increased incidence of reinfarction, death and thromboembolism early after myocardial infarction. Whether the increased platelet consumption reflects platelet deposition in the coronary vessels or in the left ventricle (as a mural thrombus) is at present under investigation with imaging of III-Indium-labeled platelets.

SummaryWe have developed a sensitive and quantitative method of “ex vivo” evaluation of platelet deposition on collagen strips, from rabbit Achilles tendon, superfused by flowing blood and applied it to four animal species, cat, rabbit, dog and pig. Autologous platelets were labeled with indium-111-tropolone, injected to the animal 24 hr before the superfusion and the number of deposited platelets was quantitated from the tendon gamma-radiation and the blood platelet count.We detected some platelet consumption with superfusion time when blood was reinfused entering the contralateral jugular vein after collagen contact but not if blood was discarded after the contact. Therefore, in order to have a more physiological animal model we decided to discard blood after superfusion of the tendon.In all species except for the cat there was a linear relationship between increase of platelet on the tendon and time of exposure to blood superfusion. The highest number of platelets deposited on the collagen was found in cats, the lowest in dogs.Ultrastructural analysis showed the platelets were deposited as aggregates after only 5 min of superfusion.

Background —Flavone-8-acetic acid (FAA; [Flavonoid]), an adjuvant antitumor drug, inhibits ristocetin-induced aggregation of human platelets. The effect of FAA on platelet-dependent thrombosis was studied in vivo in the porcine carotid artery after deep arterial injury by balloon angioplasty. Methods and Results — 111 In-labeled autologous platelet and 125 I-labeled porcine fibrin(ogen) deposition, and the incidence of macroscopic mural thrombosis onto deeply injured artery (tunica media) were compared in 20 pigs (40±1 kg [mean±SEM], body surface area=1.0±0.1 m 2 ), randomized to FAA bolus (n=10) of 5.5g/m 2 , followed by an infusion at 0.14g · m −2 · min −1 or placebo (n=10). Vasoconstriction was measured immediately beyond the dilated segment using quantitative angiography. Platelet deposition (×10 6 /cm 2 of carotid artery) was reduced over 12-fold in pigs treated with FAA (13±3 versus 164±51, P =0.001) compared with placebo. Fibrin(ogen) deposition (×10 12 molecules/cm 2 of carotid artery) did not significantly differ in FAA-treated pigs versus placebo (40±8 versus 140±69, P =0.08). Large mural thrombi were present in 100% of placebo-treated pigs versus very small thrombi in 40% of FAA-treated pigs ( P =0.005). Vasoconstriction was reduced from 46±6% in the placebo group to 15±3% in the FAA group ( P <0.001). Plasma level of FAA before angioplasty was 515±23 μg/mL. The activated partial thromboplastin time was unchanged. The bleeding time was >2SD above the normal mean in 4 of 5 treated pigs (increased from 157±29 to 522±123 s). Conclusions —FAA markedly reduced platelet deposition, mural thrombi, and injury-induced vasoconstriction after deep arterial injury, suggesting that a major inhibition of platelet glycoprotein Ibα may be beneficial therapy.

Background Thrombin plays an important role in the pathogenesis of acute coronary thrombosis. We studied the effects of a direct thrombin inhibitor, recombinant desulfatohirudin, and heparin on plasma levels (at 0, 4, 12, and 24 hours) of fibrinopeptide A (FPA), which reflects thrombin action, and prothrombin fragment F1.2, which reflects thrombin generation, in patients with unstable angina. Methods and Results Patients were randomized to one of two doses of heparin (n=50) (target activated partial thromboplastin time, 65 to 90 seconds or 90 to 110 seconds) or one of four doses of r-hirudin (n=113) (0.05, 0.10, 0.20, or 0.30 mg·kg −1 ·h −1 by infusion). r-Hirudin induced a dose-dependent decline in plasma FPA. At 24 hours, FPA levels with 0.1- to 0.3-mg·kg −1 ·h −1 r-hirudin regimens were significantly lower than with 0.05 mg·kg −1 ·h −1 r-hirudin; levels with 0.1- to 0.2-mg·kg −1 ·h −1 r-hirudin regimens were lower than with both heparin regimens. Plasma F1.2 did not decline significantly during therapy with heparin or hirudin except at 0.3 mg·kg −1 ·h −1 hirudin. At 24 hours, they were higher with the 0.05-mg·kg −1 ·h −1 r-hirudin regimen than with other regimens. For comparable levels of thrombin generation (F1.2 levels), FPA levels were higher in heparin patients than in hirudin patients. For the same FPA values, the corresponding F1.2 values were higher in the hirudin group. Conclusions Our findings provide evidence for distinct in vivo effects of the two agents and suggest that r-hirudin is a relatively more potent inhibitor of thrombin action but a less effective inhibitor of thrombin generation than heparin. The lower FPA levels in hirudin patients may reflect its ability to inactivate clot-bound thrombin. The relative clinical efficacies of the two agents need to be defined by clinical trials in progress.

Background Thrombolysis to normal flow in patients with acute myocardial infarction preserves left ventricular function and decreases mortality. Failure of early reperfusion, reocclusion, or residual thrombus may be due to concurrent activation of the platelet-coagulation system. Thus, we hypothesized that the best concomitant antithrombotic therapy (recombinant [r]-hirudin, heparin, or aspirin) will maximally accelerate thrombolysis by r–tissue-type plasminogen activator (rTPA) and reduce residual thrombus. Methods and Results Occlusive thrombi were formed in the carotid arteries of 29 pigs (by balloon dilatation followed by endarterectomy at the site of injury-induced vasospasm) and matured for 30 minutes before rTPA was started, with or without antithrombotic therapy. Thrombolysis was assessed with the use of angiography and measurement of residual thrombus. Pigs were allocated to one of five treatments: placebo, rTPA, rTPA plus r-hirudin, rTPA plus heparin, or rTPA plus intravenous aspirin. No placebo-treated pig reperfused. Two of six animals treated with rTPA alone reperfused compared with seven of seven animals treated with rTPA plus r-hirudin (reperfusion time, 33±10 minutes), six of seven animals treated with rTPA plus heparin (reperfusion time, 110±31 minutes), and two of six animals with rTPA plus aspirin. The activated partial thromboplastin time was prolonged in only the rTPA plus r-hirudin group (25±0.1 times baseline) and the rTPA plus heparin group (5.3±0.2 times baseline). Residual 111 In-platelet and 125 I-fibrin(ogen) depositions were lower in the heparin-treated group and lowest in the r-hirudin–treated group (heparin versus hirudin, respectively; incidence of residual macroscopic thrombus was six of six animals versus two of seven [ P =.01]; 125 I-fibrin(ogen), 170±76 versus 48±6 ×10 6 molecules/cm 2 [ P =.02]; 111 In-platelets, 47±15 versus 13±2 ×10 6 /cm 2 , P =.10). No pigs developed spontaneous bleeding. Conclusions Thrombin inhibition with heparin or r-hirudin significantly accelerated thrombolysis of occlusive platelet-rich thrombosis, but only the best antithrombotic therapy (r-hirudin) eliminated or nearly eliminated residual thrombus.

BACKGROUND Hypercholesterolemia is characterized by a coronary vasoconstrictive response to the endothelium-dependent vasodilator acetylcholine. This abnormality may be due to reduced synthesis of endothelium-derived relaxing factor and/or enhanced synthesis and release of an endothelium-derived contracting factor. Endothelin is an endothelium-derived vasoconstrictor and mitogenic peptide that is present in normal plasma, and its circulating concentrations are elevated in disease states that are characterized by abnormal endothelium-dependent relaxation to acetylcholine. The current studies were designed to test the hypotheses that experimental hypercholesterolemia results in elevation of plasma and tissue endothelin immunoreactivity and that the abnormal acetylcholine-evoked coronary vasoconstriction in the hypercholesterolemic animals is associated with further elevation of plasma endothelin. METHODS AND RESULTS Plasma concentrations and molecular forms of endothelin immunoreactivity were determined following 2% cholesterol diet for 4 months in pigs and during intracoronary acetylcholine administration. Second, we assessed the presence of endothelin in the coronary vascular wall by using immunohistochemistry. Hypercholesterolemia elevated plasma endothelin concentration and enhanced coronary artery tissue endothelin immunoreactivity. The endothelium-dependent vasodilator acetylcholine further increases plasma endothelin in hypercholesterolemia in association with coronary vasoconstriction. The predominant molecular form of endothelin in hypercholesterolemia is the biological active endothelin-1. CONCLUSIONS This study suggests a role for endothelin as an early participant and a marker for the endothelial dysfunction in hypercholesterolemia as well as a participant in the atherogenic process.

BACKGROUND Because the specific antithrombin hirudin prevents platelet-rich arterial thrombus and accelerates thrombolysis in a variety of animal models, it has promise as antithrombotic therapy. We therefore studied the half-life, effect on anticoagulant parameters, and safety of hirudin in patients with coronary artery disease. METHODS AND RESULTS Thirty-eight men and 1 woman (age [mean +/- SD], 60.4 +/- 6.9 years) with angiographic coronary disease were allocated in a single-blind ascending dosage study to a 6-hour i.v. infusion of recombinant hirudin (CGP 39,393) or matching placebo. The median terminal half-life for hirudin, measured by ELISA, was 2.7, 2.3, 2.9, 3.1, and 2.0 hours for the 0.02, 0.05, 0.1, 0.2, and 0.3 mg.kg-1 x h-1 groups, respectively. Activated partial thromboplastin times (aPTT) at 3, 4, and 6 hours were averaged into a plateau value. The aPTT plateau-to-baseline ratios were 1.5 +/- 0.1, 2.0 +/- 0.1, 2.3 +/- 0.1, 2.7 +/- 0.1, and 2.9 +/- 0.1, respectively, with hirudin infused at 0.02, 0.05, 0.1, 0.2, and 0.3 mg.kg-1 x h-1. From 62% to 77% of the aPTT plateau value was seen within 30 minutes of starting the infusions and was directly related to dose. The aPTT-to-baseline ratios correlated well with plasma hirudin levels (r = .88), whereas poor correlation and sensitivity were observed between plasma hirudin levels and activated coagulation time (ACT)-to-baseline ratios (r = .44). Plasma levels of hirudin and ACT in seconds correlated overall well (r = .80), but considerable overlap occurred between baseline ACT and ACT at plasma hirudin concentrations < 1000 ng/mL. Prothrombin times were significantly prolonged only at a dosage of > or = 0.05 mg.kg-1 x h-1 and were 11.8 +/- 0.5 (INR = 1.0), 12.3 +/- 0.7 (INR = 1.1), 13.3 +/- 1.2 (INR = 1.4), 14.2 +/- 0.4 (INR = 1.7), and 15.8 +/- 0.9 (INR = 2.3) seconds for each respective hirudin dosage. Thrombin times were beyond range (> 600 seconds) at 6 hours in all except 2 patients who received the lowest dosage. All parameters returned to baseline between 8 and 18 hours after the infusion. Bleeding times were not significantly prolonged. No side effects occurred. No antibodies to hirudin were detected 2 weeks after the infusion. CONCLUSIONS Recombinant hirudin has a terminal half-life of 2 to 3 hours. The aPTT correlates well with plasma levels of hirudin and allows close titration over a wide range of anticoagulation, while ACT and prothrombin time are relatively insensitive for monitoring hirudin administration. At anticoagulant levels effective in experimental thrombosis, a 6-hour infusion of hirudin is well tolerated and safe in a predominantly male group of patients with stable coronary atherosclerosis.