Didisheim, Paul

Platelets have been labeled with a new, neutral, lipidsoluble metal-complex of Indium-111-tropolone (In-TPL). Unlike oxine, tropolone is soluble in isotonic saline. Oil/saline partition of In-111-oxine and In-TPL are 0.5 and 23.7 respectively. Platelet labeling with In-TPL was performed in both ACD-plasma and ACD-saline media within two hours' time. Increasing concentration of tropolone, citrate ion, and plasma proteins decreases plateletlabeling efficiency. Effects of pH, temperature, platelet concentration, and calcium ion concentration on platelet labeling were studied. In optimum platelet-labeling conditions, canine, rabbit, porcine, and human platelets have been labeled with a consistent labeling efficiency of 80-90%. The optimum labeling conditions are In-TPL in ACD- saline and ACD-plasma at tropolone concentration of 5 and 10 μg/ml respectively and 30 minutes’ incubation of platelets with the tracer at room temperature. A kit formulation for convenient routine preparation of In-111-labeled platelets has been developed. The function of the In-TPL- labeled platelets has been studied by their aggregability with adenosine diphosphate. The capacity of In-111-labeled platelets to aggregate in vitro does not correlate well with their ability to circulate in vivo. No adverse effect of In-TPL on platelets has been observed by studies of biodistribution, recovery, and platelet survival in dogs. Comparable mean platelet survival times of four repeated studies in five dogs were obtained in both ACD-saline and ACD-plasma. These results indicate that Indium-111 platelets labeled by tropolone carrier may be preferable to oxine carrier for studies of platelet survival, imaging sites of endothelial damage, and thrombus formation.

Subcellular distribution of In-111 in In-111-labeled platelets (In-P1) determines the biodistribution and dosimetry of In-111 after the in vivo lysis of In-P1. Human platelets were labeled with In-111-oxine (In-Ox) and In- 111-troPolone (In-TPL) in ACD/saline (A/S) and plasma (P). In-P1 was lysed after 6 repeated freezing and thawing procedures. A fraction of In-P1-lysate (In-P1-LS) was filtered (0.22 ym) and passed through a calibrated Sephadex G-100 column for estimation of molecular weight (Mol. wt.) of In-bound Pl-protein. The In-P1-LS was injected in 15 rabbits and biodistribution (% I.D.) performed at 24 hours after I.V. administration. Results are tabulated below: 80 ± 11% and 70 ± 10% of In was bound to soluble platelet protein when platelets were labeled in plasma and A/S respectively. Mol. wt. of In-bound platelet protein was estimated as 52,000 ± 5,000 daltons. Biodistribution shows that In-PI-LS labeled in A/S localizes more in RES than plasma; whole body retention is also longer. These studies shed further light on the binding sites of In in platelets labeled in A/S or plasma media and their ultimate fate after administration

To quantitatively study platelet deposition on arterial prostheses and the effect of platelet inhibitory therapy with ticlopidine, 14 dogs underwent bilateral femoral artery excision and replacement with one polytetrafluoroethylene (Gore-Tex) and one autologous vein graft. Five dogs received 60 mg/kg/day of ticlopidine orally on four consecutive days prior to surgery. Nine untreated dogs served as controls. Collagen-induced platelet aggregometry studies were performed at the end of day 4. Autologous indium-111-labeled platelets were injected 24 hours prior to surgery to serve as aplatelet marker. All animals received heparin (1 mg/kg I.V.) 5 minutes prior to excision of the arterial segment. Grafts were removed 1 hour following resumption of blood flow and gently flushed with 30 cc normal saline. Radioactivity per unit weight of Gore-Tex, vein and blood was determined with a gamma counter. The relative radioactivity with respect to the internal reference standard, blood, is tabulated below.Ticlopidine treated dogs showed a 15-fold reduction in platelet deposition on Gore-Tex grafts and a 3-fold reduction on venous grafts. Platelet deposition was reduced significantly in Gore-Tex grafts (P<0.001) to a level comparable to that in autologous veins. Platelet inhibitory effect was also observed in collagen-induced platelet aggregometry.According to this “in vivo” data, ticlopidine appears to be a promising platelet inhibiting agent.

The variability of normal ranges in different laboratories and the occurrence of occasional elevated values of β-TG and PF-4 in normal subjects are well established. We examined the influence of factors such as age, sex, smoking, exercise, oral contraceptives and day-to-day variation in 148 healthy subjects. The β-TG and PF-4 assays were performed by commercial RIA’s modified by utilizing an EDTA- PGE1 -theophylline mixture as anticoagulant.The statistical method used to compare our groups was a student t-test after log transformation of the data. Our baseline control group (N=63) were non smokers and not taking oral contraceptives (O/C) or any medications. There was no difference between sexes or a significant change with increasing age, although the mean β-TG and PF-4 does increase in cohosts of 20 years. [<30 years (21.65±8.35)(SD), 30-50 years (24.32±10.94) >50 years (27.88±14.3)-β-TG data].Women on O/C were significantly different from those not on O/C for β-TG [35.06±22.45 (O/C) and 23.67±9.38 (N)] but not for PF-4 values [3.79±1.77 (O/C) and 3.91±1.63 (N)].Strenuous exercise was the parameter that influenced both β-TG and PF-4 significantly, showing an elevated value after exercise for both β-TG [81.9±31.4 v 26.73±10.91] and PF-4 [7.08±1.71 v 3.73±1.91].Day-to-day variations in β-TG and PF-4 were examined in 10 subjects for 7-10 days. The coefficient of variation (CV) amongst these subjects was 21-51%. In athletes day-today variation was the same unless a change in pattern of exercise occurred [CV-49-132%].Our results confirm that parameters such as above do influence normal values and should be taken into consideration in establishing a normal range.It also indicates the β-TG is a more sensitive parameter for delineating differences in physiological parameters in normal subjects than PF-4, and this may be applicable to clinical studies.

The skin bleeding time (BT)(Mielke, Ivy, Duke) is an important part of the diagnostic workup of bleeding disorders. Because of variable vascularity of the skin from one location to another, results may vary widely with the site and type of incision and the thickness of the skin. In some patients bleeding is difficult to stop. The discomfort of the test and the possibility of inducing scarring limit the frequency with which it can be repeated. A standardized BT that could be performed on the patient’s blood in the laboratory could avoid these problems. We have developed such a test, basing it on our previously described method in which a hole is made in a Teflon arteriovenous shunt in rats, and modified from the method of Blakely et al. A cylindrical hole of 75 μ diameter is made in a 50 cm length of 457 μ I.D. Teflon tubing with the aid of a needle, micromanipulator and dissecting microscope. Blood (2 U heparin/ml) from normal human subjects displaces buffer from the tubing at a rate (.6 ml/min) determined by a syringe pump. Pressure is controlled by raising the distal end of the tubing 5 cm above the hole. BT is recorded by an electronic drop rate counter (Payton BTR 400). Under these conditions, BT is 150 ± 47 sec, and bleeding rate from the hole, shear rate and shear stress on the wall of the hole are .11 ± .02 ml/ min, 40,700 ± 8,100 sec-1 and 1,830 ± 360 dynes/cm2 respectively. The BT can be varied from 0 to >15 min by lowering or raising pressure or flow in the system or by varying hole diameter. By phase microscopy the hole is seen to be occluded by a mass of aggregated platelets and occasional PMN. These observations show that hemostasis at small incisions is controlled by rheologic factors and platelets and does not require physical or chemical properties of a blood vessel. This new method may be useful in the diagnosis of bleeding disorders and in studying the effects of rheologic factors and drugs in hemostasis.

Platelet calcium appears to be essential in platelet activation. Accordingly, Verapamil (a channel calcium blocker) was tested “in vivo” as a possible platelet inhibitor agent. Fifteen dogs underwent bilateral femoral artery grafting with one polytetrafluoroethylene (Gore-Tex) and one autologous vein graft. Peroperatively, five dogs were infused continuously with Verapamil (6 μgs/kg/min) and 9 dogs were left untreated and the arterial prostheses were surgically implanted. Using autologous Indium-111-labeled platelets injected 24 hours prior to surgery as a platelet marker, grafts were removed 1 hour following resumption of blood flow. Radioactivity per unit weight of Gore-Tex, vein and blood were determined with a gamma counter and the relative radioactivity with respect to the internal reference standard of blood is tabulated belowVerapamil-treated dogs showed a significant reduction in the “in vivo” platelet deposition on Gore-Tex and autologous venous grafts. Preliminary data indicates that “in vitro” platelet aggregation with soluble calf skin collagen (515 μg/ml) and ADP (2-100 μm) is not different between control and drug-treated dogs.Calcium channel blockers may be promising “in vivo” platelet inhibitor agents; in addition, they may provide further understanding of the different processes involved in “in vivo” and “in vitro” platelet activation.