Platelet Consumption by Arterial Prostheses: The Effects of Endothelialization and Pharmacologic Inhibition of Platelet Function LAURENCE A. HARKER, M.D., SHERRILL J. SLICHTER, M.D., LESTER R. SAUVAGE, M.D.
The thrombogenic mechanism of arterial grafts has been studied by determining the relative utilization of platelets, fibrinogen and plasminogen by human arterial prostheses, and by direct examination of arterial grafts in a baboon model. Forty-one survival and turnover measurements of 51Crplatelets, 131I-fibrinogen and 1251-plasminogen in ten patients with aortofemoral knitted Dacron prostheses demonstrated platelet consumption after graft placement (platelet survival 4.2 days + 0.5 and turnover 68,000 plat/ul/day + 10,000 compared with 8.2 days + 0.3 and 35,000 plat/ul/day + 5,000 respectively for control subjects with stable vascular disease, p < 0.01). In vitro platelet function test results were normal. Platelet consumption was interrupted by dipyridamole or a combination of dipyridamole and acetylsalicylic acid, and platelet survival normalized spontaneously during nine months postoperatively. No significantly increased consumption of fibrinogen or plasminogen was found in these patients with arterial grafts. Placement of impervious knitted Dacron velour aortic grafts in baboons reproduced platelet consumption that progressively normalized over six weeks postoperatively. Platelet survival measurements correlated directly with endothelial cell coverage of the graft luminal surface in these animals implying that endothelialization of the graft surface was also occurring postoperatively in patients. D
ESPITE MANY TECHNICAL IMPROVEMENTS, vas-
cular prostheses continue to be thrombogenic.7,36'404243 Consequently, the benefits of prosthetic vascular grafts are currently limited to patients with disease of large and medium arteries since small caliber grafts regularly become occluded. In the present study the thrombogenic character of aortic grafts and the antithrombotic efficacy of graft endoReprint requests and correspondence: Dr. L. A. Harker, Division of Hematology, Harborview Medical Center, 325-9th Ave., Seattle, Washington 98104. Supported by research grants (HL-11775, RR-00166 and HL15257) from the U.S. Public Health Service. A portion of this work was conducted through the Clinical Research Center's facility of the University of Washington with the support of grants (FR-37 and FR-133) from the National Institutes of Health. Submitted for publication: December 6, 1976.
594
From the Division of Hematology and Regional Primate Center of the University of Washington, Puget Sound Blood Center and the Reconstructive Cardiovascular Research Center of Providence Medical Center,
Seattle, Washington
thelialization and dipyridamole were investigated in both man and baboons by serial determinations of platelet, fibrinogen and plasminogen kinetics, as well as by direct examination of grafts in baboons. Methods and Materials Patient Studies Forty-one platelet, fibrinogen and plasminogen kinetic studies were carried out in ten patients (eight males and two females) one month and serially at three, six and nine or more months following placement of knitted Dacron aortoiliac, aortofemoral or aortofemoral and femoropopliteal prostheses as indicated in Figure 1. The average age was 63 years (range 47-70 years). The grafting procedures were indicated for symptomatic angiographically advanced, large vessel atherosclerotic disease. Repeat studies were carried out one month after grafting in six of these patients while receiving 100 mg oral dipyridamole four times daily, two days preceding and throughout the platelet survival study. Four of these patients were also studied two weeks later while receiving a combination of 75 mg of dipyridamole and 330 mg of ASA three times daily for two days preceding and throughout the platelet survival study. Previous studies have shown that platelet and fibrinogen survivals reach normal values by the end of the second postoperative week in patients undergoing major surgery.35 For age and disease matched controls we selected six men between the ages of 47 and 69 years (mean 63
Vol. 186
595
PLATELET CONSUMPTION
No. 5
years) with extensive but stable, angiographically demonstrated atherosclerotic disease involving aorta, iliac and femoral arteries. Baboon Studies Twenty-four normal male baboons (Papio anubis) were also studied. They weighed 10-12 kg and had hematocrits of 40% + 3, leukocyte counts of 5,100/ul + 1,200 and platelet counts of 279,000/ul + 58,000. The animals were free of tuberculosis, dewormed and free of disease for at least six weeks before use. A 15 cm length knitted Dacron prosthesis (Filamentous velour, USCI, Inc., Billerica, Mass.) rendered impervious by an outer coating of silicone elastomer was placed to bypass the aorta, extending from the proximal end of the upper descending thoracic aorta through a retroperitoneal tunnel to the side of the abdominal aorta below the renal arteries (Fig. 1). During the period of postoperative study the animals were maintained in restraining chairs to facilitate repeated blood sampling.13'19 Platelet and fibrinogen concentration and survival were measured before and serially at weekly intervals after aortic bypass grafting for up to six weeks. Sham operations in control animals demonstrated that platelet and fibrinogen survivals had normalized within one week of surgery. Three or four animals were sacrificed each week throughout the study to permit morphologic study of the grafts. Laboratory Studies Platelet counts were measured with an electronic particle counter on peripheral blood collected in EDTA as previously described.5"2 The platelet count of 100 normal subjects was 250,000 per ul + 40,000* while that of 63 normal baboons was 275,000/ul + 55,000. Platelet survival was determined by measuring the disappearance of radioactivity from blood sampled ten to 11 times after injection of autologous 51Cr-labeled platelets. 12.13.17,19
Mean platelet survival time was determined by a gamma function as described by Murphy27-29 and was 9.5 + 0.6 days in 35 young adult volunteers and 9.0 + 0.8 days for 28 older normal volunteers averaging 47 years (range 35-71). The proportion of labeled platelets remaining within the systemic circulation after infusion (i.e., "recovery") was calculated from the platelet activity per milliliter, extrapolated to zero time, multiplied by the estimated blood volume, and divided by the platelet 51Cr activity injected. Platelet consumption, measured computer fitting to
*
Variances in the methods section
are
given
as
±t1
SD.
Cuff of
I
FIG. 1. Aortic grafts in baboons. Fifteen centimeter length knitted Dacron filamentous vascular prostheses (USCI, Inc.), rendered impervious by an outer coating of silicone elastomer (Silastic, Dow Corning), were placed as thoracoabdominal bypasses in 24 baboons. Noncoated cuffs of the same material were used for the anastomoses, to avoid problems of anastomotic tearing occasioned by the stiffness of the coated tube. Platelet and fibrinogen concentration and survival were measured before aortic bypass grafting and again, serially at weekly intervals up to 6 weeks, after
filamentous vascular prosthesis
External silastic coat
Cuff of
filamentous vascular prosthesis
surgery.
BYPASS
-
BABOON
blood per day, platelet count divided by the platelet survival time in days and corrected for recovery. Platelet recovery was 65% + 5 and platelet turnover was 35,000 plat/ul/day + 4,600 in 35 normal subjects. In 24 normal male baboons platelet survival was 5.5 + 0.3 days. Platelet recovery averaged 85 + 5% and platelet turnover was 59,000 plat/ul/day + 7,000. For calculating fibrinogen turnover, the concentration of fibrinogen was estimated spectrophotometrically by a modification of Jacobsson' s method in which the optical density of thrombin-clottable protein is determined after collection on a glass rod and subsequent solution in alkaline urea.22 The fibrinogen concentration in 87 normal subjects was 2.85 mg per ml + 0.22 and 2.70 mg per ml + 0.41 in 63 normal baboons. Labeling of normal fibrinogen with 1311 from a single hepatitis-free donor was performed by the method of Takeda.17'4' Fibrinogen survival, derived from the least squares, computer determined half-time disappearance and divided by the natural logarithm of 2, averaged 5.1 + 0.2 days in 35 normal patients and 2.6 + 0.2 days in 24 normal baboons. Fibrinogen turnover, used as a measure of fibrinogen consumption was calculated from the concentration of plasma fibrinogen divided by its mean survival time. In 35 normal subjects as platelet turnover per microliter of was calculated from the peripheral
HARKER, SLICHTER AND SAUVAGE
596
fibrinogen turnover averaged 0.57 mg/ml/day + 0.05 and 1.0 + 0.10 mg/ml/day in 24 normal baboons. Oral doses of nonradioactive iodide (5 mEq twice daily) were given to patients the day before injection of radioiodine labeled fibrinogen and continued for ten days. Plasminogen levels were determined by the caseinolytic23 technique and expressed as ug/ml of plasma, calculated on the basis of specific activity of 25 CTA units per mg plasminogen.'8 Concentration averaged 105 ,ug/ml + 20 in 65 normal subjects (equivalent to 2.63 CTA units/ml + 0.51). Plasminogen for 1251 labeling was prepared from single donor plasma by one step EACA-agarose affinity chromatography as described by Deutsch and Mertz.9"8 Chloramine T was used for 1251 labeling.26 In vivo plasminogen survival and turnover were determined in the same manner as for fibrinogen. In 20 normal subjects plasminogen disappeared exponentially with a mean survival time of 2.34 days + 0.16. Plasminogen turnover, an expression of the rate of removal in vivo, was regarded as reflecting overall fibrinolytic activity in vivo. The 20 normal subjects had a mean plasminogen turnover of 45 ,ug/ml/day + 5. For all studies, the labeled platelets, fibrinogen and plasminogen were mixed before injection so that their introduction was simultaneous. Serial kinetic studies were analyzed by the T-test for paired samples. Fibrinolysis was also assessed by measuring fibrinogen-fibrin related antigen (F-FRA) levels using the rapid hemagglutination inhibition technique described by Merskey25 from samples prepared with thrombinEACA at 4°. In 56 normal subjects this measurement was consistently less than 0.5 ,tg/ml. For assessing platelet function, bleeding time (normal 4.5 min. + 1.5) was measured by the template
Ann. Surg.
November 1977
modification of the Ivy technique.'6 Platelet aggregation was estimated from changes in optical transmission of 0.02 M sodium citrated plasma at 370 with a
concentration of 300,000 platelets
per
,1.d3 Ag-
gregation was observed in one milliliter samples after the addition of ADP (0.5, 1.0, 1.5, 2.0 and 10 ,uM), thrombin (0.2 NIH units), epinephrine (25 ,g) and collagen (100 and 200 g.g of standardized suspension). Plastic equipment was used throughout; and plasma, freshly drawn, was kept at room temperature during the 30-70 minutes before testing. Platelet retention by glass bead columns was measured by the Bowie modification of the Salzman method,4 blood being drawn by two-syringe technique (plastic) without anticoagulant and immediately passed through the standard column of beads by a constant infusion pump at the rate of two milliliters per minute. The delay between blood drawing and initiation of flow through the beads was less then ten seconds. Grafts were obtained for microscopic examination at the time of animal sacrifice. In four anesthetized animals, in vivo perfusion-fixation was carried out with half-strength Karnovsky's fixative under 100 cm of hydrostatic pressure for 20 minutes.'3"19 Samples were obtained from mid-area of the proximal, central and distal segments of the graft. Specimens were subsequently placed in half-strength Karnovsky's solution for six hours at 40 prepared for scanning EM, and then post fixed with osmium tetroxide, embedded in epon, and cut to one micron thickness. Sections were mounted and stained with a polychromatic dye consisting of azure II-methylene blue-basic fuchsin for light microscopic examination.20 They were subsequently processed for transmission EM. In the remaining 20 animals pressure perfusion fixation was carried out with 0.3% silver nitrate solution.3' The
TABLE 1. Effect of Antiplatelet Therapy on Aortic Graft-Related Platelet Consumption
Platelet consumption one month post grafting
*
Platelet turnover
(days)
(plat/ul/day) Combination dipyridamole 75 mg and ASA 330 mg tid
Baseline
Dipyridamole 100 mg qid
4.1 4.3 3.7 4.3 3.6 4.9
7.0 7.1 7.7 8.5 8.1 8.3
7.7 7.4
56,000 77,000 80,000
8.4 7.9
7.8* ± 0.6
7.9* ± 0.4
1 2 3 4 5 6
Mean
Platelet survival
4.2
+
0.5
Significantly different from baseline
Baseline 57,000
at p < 0.001.
Dipyridamole 100 mg qid
Combination dipyridamole 75 mg and ASA 330 mg tid
66,000 71,000
21,000 30,000 28,000 38,000 33,000 40,000
41,000 29,000
68,000 ± 10,000
32,000* ± 7,000
34,000* ± 5,000
32,000 34,000
Vol. 186 .No. 5
597
PLATELET CONSUMPTION
entire endovascular surface of whole graft mounts were examined morphometrically with a grid micrometer to assess the proportion and distribution of endothelialized surface. The grafts were subsequently fixed in ten per cent buffered formalin and prepared with paraffin embedding for conventional light microscopy using H and E staining.
I.,.,.,.,.,.':............................................................................................................................................... ". ...
9'
.. . . . . . ..............................................................
///////////////////A 6-
Results Patient Studies Mean platelet survival was shortened in all patients studied within one month after placement of aortofemoral prosthesis (mean 4.2 + 0.5 days compared with 8.2 + 0.3 days in comparably aged and diseased patients, p < 0.001, and 9.5 days + 0.6 in young normal subjects, Tables 1 and 2). The two shortest initial platelet survival times measured were in the patients with both aortofemoral and femoropopliteal grafts (Table 2). Platelet turnover was reciprocally increased two-fold (68,000 plat/,ul/day + 10,000 compared with 35,000 plat/,ul/day + 4,500 in control subjects, p < 0.01). The results of the corresponding fibrinogen survival and turnover measurements were 4.8 ± 0.3 days and 0.67 mg/ml/day + 0.10, which did not differ from the unoperated control group of patients with comparable peripheral vascular disease, i.e. 4.9 + 0.1 days and 0.63 mg/ml/day + 0.04 respectively). Plasminogen survival and turnover were 2.10 days + 0.09 and 55 ,ug/ml/day ± 6; these results were also not different from the atherosclerotic control group, i.e. 2.15 days + 0.11 and 50 ug/ml/day ± 4 respectively. The levels of F-FRA were consistently < 0.5 pig/ml. The battery of platelet function tests was carried out in all patients during the first postoperative month and at least once during the next TABLE 2. Serial Platelet Survival in Patients Following Aorto-Femoral Prothesis Placement
Patient number and proceduret 1 lb 2 la 3 lc 4 lb 5 Ic 6 la 7 la 8 lb 9 lb 10 la Mean
1 Month
3 Months
6 Months
9 Months or more
4.1 4.3 3.7 4.2 3.6 4.9 4.4
5.7 5.8 5.5
7.1 6.8 7.8 8.3
9.0 8.0 8.3 10.2
-
8.6 9.0
4.2*
+
5.4 5.2 6.8 7.7 8.5 5.3 7.9 0.5 5.5* + 0.3 7.4* + 0.6 8.7* + 0.8
* Significantly different from other values at p < 0.01. t Type of Surgical procedure identified in Fig. 1. la bifurcation aortoiliac graft. lb aortofemoral graft. Ic aortofemoral and femoropopliteal graft.
U1)
q)
3-
6 3 Time post grafting (months) FIG. 2. Platelet survival measurements in patients with arterial grafts. Platelet survival was consistently reduced one month after surgery and spontaneously normalized during the following nine months as shown by the solid circles (±1 SD). Platelet consumption was interrupted by dipyridamole (open circle) or a combination of dipyridamole and ASA (closed triangle). Baseline platelet survival times in normal subjects (B) and control patients (A) with severe but stable peripheral vascular disease (±1 SD) are shown.
six months including aggregation to ADP, epinephrine or collagen and glass bead retention. In no instance was there significant difference from control studies during the nine month postoperative period. The administration of oral dipyridamole, 100 mg qid, for one week, to six patients one month post grafting uniformly corrected platelet survival and turnover to near control values (7.8 + 0.6 days and 32,000 plat/,ullday ± 7,000, the change was significant at p < 0.001). Comparable correction of platelet survival and turnover was achieved in four patients receiving 75 mg dipyridamole and 330 mg ASA given as a combination three times daily for one week (Table 1, Fig. 2, p < 0.001). Two additional patients, studied within the first week postoperatively while receiving daily low molecular weight dextran infusions (500 ml dextran 40), had platelet survival times of 7.8 and 8.3 days. Repeat platelet survival one week after discontinuing dextran was 4.0 and 4.3 days respectively. Platelet survival and turnover were found to normalize progressively when studied serially at three, six and nine months postoperatively (Table 2, Fig. 2). Fibrinogen and plasminogen kinetics remained unchanged from baseline and control values.
Ann. Surg. a November 1977 HARKER, SLII[CHTER AND SAUVAGE TABLE 3. Relationship Between Platelet Consumption and surgery, demonstrating the absence of any effect of the Non-endothelialized Luminal Surface of on platelet survival at that time. In the grafted operation Aortic Prosthesis in Baboons group fibrinogen survival and turnover were not sigEndothelial nificantly altered; baseline values of 2.6 days ± 0.2 cell coverage 1.0 mg/ml/day + 0.1 compared with 2.4 days ± 0.4 and of prosthetic Platelet Platelet surface turnover survival No. of Time and 1.2 mg/ml/day + 0.3 one week postoperatively. (%) (days) animals (weeks) (plat/,Ulday) Both platelet survival and turnover progressively 100 5.5 + 0.2 59,000 + 7,000 24 pregrafting normalized over the subsequent five weeks (Table 3,
598
postgrafting 0 1 2 3 4 5 6 *
2 14 9 6
2.3* 2.7* 3.3* 4.0* 5.2 5.3
9 4 4
+ 0.3 ± 0.3 + 0.4 0.3 +0.3 ±0.2
Fig. 3).
153,000* + 141,000* ± 110,000* ± 91,000* 72,000 + 66,000 +
Significantly different from pregrafting values at
p
The thrombogenic mechanism of arterial grafts has been studied by determining the relative utilization of platelets, fibrinogen and plasminogen by human arterial prostheses, and by direct examination of arterial grafts in a baboon model. Forty-one survival and turnover measurements of 51Crplatelets, 131I-fibrinogen and 1251-plasminogen in ten patients with aortofemoral knitted Dacron prostheses demonstrated platelet consumption after graft placement (platelet survival 4.2 days + 0.5 and turnover 68,000 plat/ul/day + 10,000 compared with 8.2 days + 0.3 and 35,000 plat/ul/day + 5,000 respectively for control subjects with stable vascular disease, p < 0.01). In vitro platelet function test results were normal. Platelet consumption was interrupted by dipyridamole or a combination of dipyridamole and acetylsalicylic acid, and platelet survival normalized spontaneously during nine months postoperatively. No significantly increased consumption of fibrinogen or plasminogen was found in these patients with arterial grafts. Placement of impervious knitted Dacron velour aortic grafts in baboons reproduced platelet consumption that progressively normalized over six weeks postoperatively. Platelet survival measurements correlated directly with endothelial cell coverage of the graft luminal surface in these animals implying that endothelialization of the graft surface was also occurring postoperatively in patients. D
ESPITE MANY TECHNICAL IMPROVEMENTS, vas-
cular prostheses continue to be thrombogenic.7,36'404243 Consequently, the benefits of prosthetic vascular grafts are currently limited to patients with disease of large and medium arteries since small caliber grafts regularly become occluded. In the present study the thrombogenic character of aortic grafts and the antithrombotic efficacy of graft endoReprint requests and correspondence: Dr. L. A. Harker, Division of Hematology, Harborview Medical Center, 325-9th Ave., Seattle, Washington 98104. Supported by research grants (HL-11775, RR-00166 and HL15257) from the U.S. Public Health Service. A portion of this work was conducted through the Clinical Research Center's facility of the University of Washington with the support of grants (FR-37 and FR-133) from the National Institutes of Health. Submitted for publication: December 6, 1976.
594
From the Division of Hematology and Regional Primate Center of the University of Washington, Puget Sound Blood Center and the Reconstructive Cardiovascular Research Center of Providence Medical Center,
Seattle, Washington
thelialization and dipyridamole were investigated in both man and baboons by serial determinations of platelet, fibrinogen and plasminogen kinetics, as well as by direct examination of grafts in baboons. Methods and Materials Patient Studies Forty-one platelet, fibrinogen and plasminogen kinetic studies were carried out in ten patients (eight males and two females) one month and serially at three, six and nine or more months following placement of knitted Dacron aortoiliac, aortofemoral or aortofemoral and femoropopliteal prostheses as indicated in Figure 1. The average age was 63 years (range 47-70 years). The grafting procedures were indicated for symptomatic angiographically advanced, large vessel atherosclerotic disease. Repeat studies were carried out one month after grafting in six of these patients while receiving 100 mg oral dipyridamole four times daily, two days preceding and throughout the platelet survival study. Four of these patients were also studied two weeks later while receiving a combination of 75 mg of dipyridamole and 330 mg of ASA three times daily for two days preceding and throughout the platelet survival study. Previous studies have shown that platelet and fibrinogen survivals reach normal values by the end of the second postoperative week in patients undergoing major surgery.35 For age and disease matched controls we selected six men between the ages of 47 and 69 years (mean 63
Vol. 186
595
PLATELET CONSUMPTION
No. 5
years) with extensive but stable, angiographically demonstrated atherosclerotic disease involving aorta, iliac and femoral arteries. Baboon Studies Twenty-four normal male baboons (Papio anubis) were also studied. They weighed 10-12 kg and had hematocrits of 40% + 3, leukocyte counts of 5,100/ul + 1,200 and platelet counts of 279,000/ul + 58,000. The animals were free of tuberculosis, dewormed and free of disease for at least six weeks before use. A 15 cm length knitted Dacron prosthesis (Filamentous velour, USCI, Inc., Billerica, Mass.) rendered impervious by an outer coating of silicone elastomer was placed to bypass the aorta, extending from the proximal end of the upper descending thoracic aorta through a retroperitoneal tunnel to the side of the abdominal aorta below the renal arteries (Fig. 1). During the period of postoperative study the animals were maintained in restraining chairs to facilitate repeated blood sampling.13'19 Platelet and fibrinogen concentration and survival were measured before and serially at weekly intervals after aortic bypass grafting for up to six weeks. Sham operations in control animals demonstrated that platelet and fibrinogen survivals had normalized within one week of surgery. Three or four animals were sacrificed each week throughout the study to permit morphologic study of the grafts. Laboratory Studies Platelet counts were measured with an electronic particle counter on peripheral blood collected in EDTA as previously described.5"2 The platelet count of 100 normal subjects was 250,000 per ul + 40,000* while that of 63 normal baboons was 275,000/ul + 55,000. Platelet survival was determined by measuring the disappearance of radioactivity from blood sampled ten to 11 times after injection of autologous 51Cr-labeled platelets. 12.13.17,19
Mean platelet survival time was determined by a gamma function as described by Murphy27-29 and was 9.5 + 0.6 days in 35 young adult volunteers and 9.0 + 0.8 days for 28 older normal volunteers averaging 47 years (range 35-71). The proportion of labeled platelets remaining within the systemic circulation after infusion (i.e., "recovery") was calculated from the platelet activity per milliliter, extrapolated to zero time, multiplied by the estimated blood volume, and divided by the platelet 51Cr activity injected. Platelet consumption, measured computer fitting to
*
Variances in the methods section
are
given
as
±t1
SD.
Cuff of
I
FIG. 1. Aortic grafts in baboons. Fifteen centimeter length knitted Dacron filamentous vascular prostheses (USCI, Inc.), rendered impervious by an outer coating of silicone elastomer (Silastic, Dow Corning), were placed as thoracoabdominal bypasses in 24 baboons. Noncoated cuffs of the same material were used for the anastomoses, to avoid problems of anastomotic tearing occasioned by the stiffness of the coated tube. Platelet and fibrinogen concentration and survival were measured before aortic bypass grafting and again, serially at weekly intervals up to 6 weeks, after
filamentous vascular prosthesis
External silastic coat
Cuff of
filamentous vascular prosthesis
surgery.
BYPASS
-
BABOON
blood per day, platelet count divided by the platelet survival time in days and corrected for recovery. Platelet recovery was 65% + 5 and platelet turnover was 35,000 plat/ul/day + 4,600 in 35 normal subjects. In 24 normal male baboons platelet survival was 5.5 + 0.3 days. Platelet recovery averaged 85 + 5% and platelet turnover was 59,000 plat/ul/day + 7,000. For calculating fibrinogen turnover, the concentration of fibrinogen was estimated spectrophotometrically by a modification of Jacobsson' s method in which the optical density of thrombin-clottable protein is determined after collection on a glass rod and subsequent solution in alkaline urea.22 The fibrinogen concentration in 87 normal subjects was 2.85 mg per ml + 0.22 and 2.70 mg per ml + 0.41 in 63 normal baboons. Labeling of normal fibrinogen with 1311 from a single hepatitis-free donor was performed by the method of Takeda.17'4' Fibrinogen survival, derived from the least squares, computer determined half-time disappearance and divided by the natural logarithm of 2, averaged 5.1 + 0.2 days in 35 normal patients and 2.6 + 0.2 days in 24 normal baboons. Fibrinogen turnover, used as a measure of fibrinogen consumption was calculated from the concentration of plasma fibrinogen divided by its mean survival time. In 35 normal subjects as platelet turnover per microliter of was calculated from the peripheral
HARKER, SLICHTER AND SAUVAGE
596
fibrinogen turnover averaged 0.57 mg/ml/day + 0.05 and 1.0 + 0.10 mg/ml/day in 24 normal baboons. Oral doses of nonradioactive iodide (5 mEq twice daily) were given to patients the day before injection of radioiodine labeled fibrinogen and continued for ten days. Plasminogen levels were determined by the caseinolytic23 technique and expressed as ug/ml of plasma, calculated on the basis of specific activity of 25 CTA units per mg plasminogen.'8 Concentration averaged 105 ,ug/ml + 20 in 65 normal subjects (equivalent to 2.63 CTA units/ml + 0.51). Plasminogen for 1251 labeling was prepared from single donor plasma by one step EACA-agarose affinity chromatography as described by Deutsch and Mertz.9"8 Chloramine T was used for 1251 labeling.26 In vivo plasminogen survival and turnover were determined in the same manner as for fibrinogen. In 20 normal subjects plasminogen disappeared exponentially with a mean survival time of 2.34 days + 0.16. Plasminogen turnover, an expression of the rate of removal in vivo, was regarded as reflecting overall fibrinolytic activity in vivo. The 20 normal subjects had a mean plasminogen turnover of 45 ,ug/ml/day + 5. For all studies, the labeled platelets, fibrinogen and plasminogen were mixed before injection so that their introduction was simultaneous. Serial kinetic studies were analyzed by the T-test for paired samples. Fibrinolysis was also assessed by measuring fibrinogen-fibrin related antigen (F-FRA) levels using the rapid hemagglutination inhibition technique described by Merskey25 from samples prepared with thrombinEACA at 4°. In 56 normal subjects this measurement was consistently less than 0.5 ,tg/ml. For assessing platelet function, bleeding time (normal 4.5 min. + 1.5) was measured by the template
Ann. Surg.
November 1977
modification of the Ivy technique.'6 Platelet aggregation was estimated from changes in optical transmission of 0.02 M sodium citrated plasma at 370 with a
concentration of 300,000 platelets
per
,1.d3 Ag-
gregation was observed in one milliliter samples after the addition of ADP (0.5, 1.0, 1.5, 2.0 and 10 ,uM), thrombin (0.2 NIH units), epinephrine (25 ,g) and collagen (100 and 200 g.g of standardized suspension). Plastic equipment was used throughout; and plasma, freshly drawn, was kept at room temperature during the 30-70 minutes before testing. Platelet retention by glass bead columns was measured by the Bowie modification of the Salzman method,4 blood being drawn by two-syringe technique (plastic) without anticoagulant and immediately passed through the standard column of beads by a constant infusion pump at the rate of two milliliters per minute. The delay between blood drawing and initiation of flow through the beads was less then ten seconds. Grafts were obtained for microscopic examination at the time of animal sacrifice. In four anesthetized animals, in vivo perfusion-fixation was carried out with half-strength Karnovsky's fixative under 100 cm of hydrostatic pressure for 20 minutes.'3"19 Samples were obtained from mid-area of the proximal, central and distal segments of the graft. Specimens were subsequently placed in half-strength Karnovsky's solution for six hours at 40 prepared for scanning EM, and then post fixed with osmium tetroxide, embedded in epon, and cut to one micron thickness. Sections were mounted and stained with a polychromatic dye consisting of azure II-methylene blue-basic fuchsin for light microscopic examination.20 They were subsequently processed for transmission EM. In the remaining 20 animals pressure perfusion fixation was carried out with 0.3% silver nitrate solution.3' The
TABLE 1. Effect of Antiplatelet Therapy on Aortic Graft-Related Platelet Consumption
Platelet consumption one month post grafting
*
Platelet turnover
(days)
(plat/ul/day) Combination dipyridamole 75 mg and ASA 330 mg tid
Baseline
Dipyridamole 100 mg qid
4.1 4.3 3.7 4.3 3.6 4.9
7.0 7.1 7.7 8.5 8.1 8.3
7.7 7.4
56,000 77,000 80,000
8.4 7.9
7.8* ± 0.6
7.9* ± 0.4
1 2 3 4 5 6
Mean
Platelet survival
4.2
+
0.5
Significantly different from baseline
Baseline 57,000
at p < 0.001.
Dipyridamole 100 mg qid
Combination dipyridamole 75 mg and ASA 330 mg tid
66,000 71,000
21,000 30,000 28,000 38,000 33,000 40,000
41,000 29,000
68,000 ± 10,000
32,000* ± 7,000
34,000* ± 5,000
32,000 34,000
Vol. 186 .No. 5
597
PLATELET CONSUMPTION
entire endovascular surface of whole graft mounts were examined morphometrically with a grid micrometer to assess the proportion and distribution of endothelialized surface. The grafts were subsequently fixed in ten per cent buffered formalin and prepared with paraffin embedding for conventional light microscopy using H and E staining.
I.,.,.,.,.,.':............................................................................................................................................... ". ...
9'
.. . . . . . ..............................................................
///////////////////A 6-
Results Patient Studies Mean platelet survival was shortened in all patients studied within one month after placement of aortofemoral prosthesis (mean 4.2 + 0.5 days compared with 8.2 + 0.3 days in comparably aged and diseased patients, p < 0.001, and 9.5 days + 0.6 in young normal subjects, Tables 1 and 2). The two shortest initial platelet survival times measured were in the patients with both aortofemoral and femoropopliteal grafts (Table 2). Platelet turnover was reciprocally increased two-fold (68,000 plat/,ul/day + 10,000 compared with 35,000 plat/,ul/day + 4,500 in control subjects, p < 0.01). The results of the corresponding fibrinogen survival and turnover measurements were 4.8 ± 0.3 days and 0.67 mg/ml/day + 0.10, which did not differ from the unoperated control group of patients with comparable peripheral vascular disease, i.e. 4.9 + 0.1 days and 0.63 mg/ml/day + 0.04 respectively). Plasminogen survival and turnover were 2.10 days + 0.09 and 55 ,ug/ml/day ± 6; these results were also not different from the atherosclerotic control group, i.e. 2.15 days + 0.11 and 50 ug/ml/day ± 4 respectively. The levels of F-FRA were consistently < 0.5 pig/ml. The battery of platelet function tests was carried out in all patients during the first postoperative month and at least once during the next TABLE 2. Serial Platelet Survival in Patients Following Aorto-Femoral Prothesis Placement
Patient number and proceduret 1 lb 2 la 3 lc 4 lb 5 Ic 6 la 7 la 8 lb 9 lb 10 la Mean
1 Month
3 Months
6 Months
9 Months or more
4.1 4.3 3.7 4.2 3.6 4.9 4.4
5.7 5.8 5.5
7.1 6.8 7.8 8.3
9.0 8.0 8.3 10.2
-
8.6 9.0
4.2*
+
5.4 5.2 6.8 7.7 8.5 5.3 7.9 0.5 5.5* + 0.3 7.4* + 0.6 8.7* + 0.8
* Significantly different from other values at p < 0.01. t Type of Surgical procedure identified in Fig. 1. la bifurcation aortoiliac graft. lb aortofemoral graft. Ic aortofemoral and femoropopliteal graft.
U1)
q)
3-
6 3 Time post grafting (months) FIG. 2. Platelet survival measurements in patients with arterial grafts. Platelet survival was consistently reduced one month after surgery and spontaneously normalized during the following nine months as shown by the solid circles (±1 SD). Platelet consumption was interrupted by dipyridamole (open circle) or a combination of dipyridamole and ASA (closed triangle). Baseline platelet survival times in normal subjects (B) and control patients (A) with severe but stable peripheral vascular disease (±1 SD) are shown.
six months including aggregation to ADP, epinephrine or collagen and glass bead retention. In no instance was there significant difference from control studies during the nine month postoperative period. The administration of oral dipyridamole, 100 mg qid, for one week, to six patients one month post grafting uniformly corrected platelet survival and turnover to near control values (7.8 + 0.6 days and 32,000 plat/,ullday ± 7,000, the change was significant at p < 0.001). Comparable correction of platelet survival and turnover was achieved in four patients receiving 75 mg dipyridamole and 330 mg ASA given as a combination three times daily for one week (Table 1, Fig. 2, p < 0.001). Two additional patients, studied within the first week postoperatively while receiving daily low molecular weight dextran infusions (500 ml dextran 40), had platelet survival times of 7.8 and 8.3 days. Repeat platelet survival one week after discontinuing dextran was 4.0 and 4.3 days respectively. Platelet survival and turnover were found to normalize progressively when studied serially at three, six and nine months postoperatively (Table 2, Fig. 2). Fibrinogen and plasminogen kinetics remained unchanged from baseline and control values.
Ann. Surg. a November 1977 HARKER, SLII[CHTER AND SAUVAGE TABLE 3. Relationship Between Platelet Consumption and surgery, demonstrating the absence of any effect of the Non-endothelialized Luminal Surface of on platelet survival at that time. In the grafted operation Aortic Prosthesis in Baboons group fibrinogen survival and turnover were not sigEndothelial nificantly altered; baseline values of 2.6 days ± 0.2 cell coverage 1.0 mg/ml/day + 0.1 compared with 2.4 days ± 0.4 and of prosthetic Platelet Platelet surface turnover survival No. of Time and 1.2 mg/ml/day + 0.3 one week postoperatively. (%) (days) animals (weeks) (plat/,Ulday) Both platelet survival and turnover progressively 100 5.5 + 0.2 59,000 + 7,000 24 pregrafting normalized over the subsequent five weeks (Table 3,
598
postgrafting 0 1 2 3 4 5 6 *
2 14 9 6
2.3* 2.7* 3.3* 4.0* 5.2 5.3
9 4 4
+ 0.3 ± 0.3 + 0.4 0.3 +0.3 ±0.2
Fig. 3).
153,000* + 141,000* ± 110,000* ± 91,000* 72,000 + 66,000 +
Significantly different from pregrafting values at
p
